molecular genetics review Flashcards
Review definitions
Chromosomes: a complex arrangement of DNA
Genes: sequences of DNA that code for a particular protein (can be many genes on one chromosome)
A genome is the whole of the genetic info of an organism. In humans, the genome consists of the 46 molecules that form the chromosomes in the nucleus PLUS the DNA molecule in the mitochondria. In plants, the genome is the DNA molecules of chromosomes in the nucleus plus the DNA in the mitochondria and the chloroplast. In prokaryotes, the genome is much smaller and consists of the DNA in the circular chromosome, plus any plasmids that are present.
Diploid (2n): indicates a complete set of chromosomes. found in somatic cells (all besides sex cells)
Halpoid (n): indicates half of a complete set of chromosomes. found in sex cells (aka gametes)
The number of chromosomes doesn’t indicate the complexity of the organism
Human Chromosomes: Human diploid # 2n= 46 (i.e. 46 chromosomes in every somatic cell). Somatic cells reproduce by mitosis to produce more identical somatic cells
Homologous chromosomes: carry info for the same trait in the same position on the chromosome. From each pair – one chromosome comes from the mother (maternal) and one from the father (paternal). Humans have 23 pairs of HC. 22 pairs are autosomes that control things like hair colour, eye colour etc.. The last pair are the sex chromosomes that determine sex
Sex chromosomes: female has to X and male has XY
Karyogram – a picture of the 46 chromosomes in a human somatic cell –> Chromosomes are paired based on having similar size and position of centromeres, and ordered from largest to smallest.
Genes: A length of DNA – carries info for a specific trait. Has a particular position (or locus) on a chromosome. 2 chromosomes from a homologous pair carry genes for the same trait at the same locus. Each homologous pair of chromosomes carries genetic info for thousands of hereditary traits
DNA of prokaryotic cells: Circular, double stranded DNA. Usually only one copy of gene → haploid. Genomes carry very little non-essential DNA. Bacterial chromosomes are packed into the nucleoid region. Bacterial DNA is compacted ~1000X
Plasmids: Small, circular or linear DNA molecules. Contained within some prokaryotes. Not part of nucleoid and often carry non-essential genes. Can be copied and transmitted b/w cells or incorporated into cell’s DNA and reproduced during cell division
DNA of eukaryotic cells: More DNA than prokaryotic cells. Contained in nucleus. More compaction of DNA than in prokaryotes (achieved through different levels of organization)
Supercoiling in prophase: Chromatin condenses into shorter, thicker strands. Supercoiling – repeatedly coiling the DNA molecule to make the chromosome shorter and wider. involves histone proteins and enzymes
Eukaryotic genome can vary in chromosome number and the organization of genes can differ. The size and number of genes in the genome can vary and it doesn’t indicate complexity
Genes: Sequences of DNA that code for a particular protein (there can be many genes on one chromosome). Sections of DNA that contribute to certain traits, characteristics or functions. Genes code for proteins or parts of proteins that influence things like the immune system, skin pigmentation etc. Genes are chunks of DNA that contribute to particular traits or functions by coding for proteins that influence physiology.
Alleles: Alleles are different versions of a gene, which vary according to the nucleotide base present at a particular genome location. An individual’s combination of alleles is known as their genotype.
Gregor Mendel, through his work on pea plants, discovered the fundamental laws of inheritance. He deduced that genes come in pairs and are inherited as distinct units, one from each parent. Mendel tracked the segregation of parental genes and their appearance in the offspring as dominant or recessive traits. He recognized the mathematical patterns of inheritance from one generation to the next. Mendel’s Laws of Heredity are usually stated as:
1) The Law of Segregation: Each inherited trait is defined by a gene pair. Parental genes are randomly separated to the sex cells so that sex cells contain only one gene of the pair. Offspring therefore inherit one genetic allele from each parent when sex cells unite in fertilization.
2) The Law of Independent Assortment: Genes for different traits are sorted separately from one another so that the inheritance of one trait is not dependent on the inheritance of another.
3) The Law of Dominance: An organism with alternate forms of a gene will express the form that is dominant.
A mutation is a change in the DNA sequence of an organism. Mutations can result from errors in DNA replication during cell division, exposure to mutagens or a viral infection.
Chromatin is a long chain of DNA. Chromosomes is rolled up DNA when it is going through cell division. Sister chromatids are the branches of the same chromosome. sister chromatids are genetically identical, whereas homologous chromosomes are composed of two different chromosomes that are not genetically identical despite containing the same sets of genes. The sister chromatids are joined to each other via the centromere and only become separated when cell division occurs. Homologous chromosomes, by contrast, are pairs of genetically distinct chromosomes. While each pair codes for similar genetic traits, they are genetically non-identical.
DNA Structure and Cellular Organization Summary:
Linus Pauling and protein helical structure:
- First developed methods of assembling 3D models using known distances and bond angles between atoms and molecules
- 1951 – many proteins have helix-shaped structures
- Crick hypothesized that DNA may also have a helical structure
Franklin and DNA helical structure:
- Franklin used X-ray diffraction to analyze the structure of biological molecules
- Purified substance is subjected to X-rays
- Bending (diffraction) by X-rays due to presence of molecules can be visualized
- Mathematical theory is used to interpret these photos
Watson and Crick build 3D DNA model:
- Watson and Crick deduced that DNA is a twisted, ladder-like structure, called a double helix.
- Sugar-phosphates make up the sides
- Bases make up the ladder’s rungs
- Franklins images showed the distance between sugar–phosphate handrails equal
- Chargaff’s rule inferred A – T or C – G base pairing.
The double helix Modern DNA model:
- 2 Polynucleotide strands that twist around each other to form a double helix
- 2 Strands of a DNA molecule are not matching but complementary, due to complementary base pairing
- Hydrogen bonds link base pairs
- Strands are antiparallel, and therefore ‘face’ in opposite directions 3’-5’, 5’-3’
- The 5’ and 3’ designations refer to the number of the C atom in a deoxyribose sugar molecule to which the phosphate and hydroxyl groups bond, respectively
Structure & Organization of Genetic Material in Prokaryotes and Eukaryotes:
- Genetic Cellular organization: Primary structure of DNA is sequence of nucleotides. Secondary is the strands coming together.
- Total genetic material of an organism is genome while genes are the functional units of DNA. There are noncoding and coding regions of DNA.
DNA of Prokaryotic Cells:
- Genetic material is in the form of a circular, double-stranded DNA molecule.
- May have more than one copy present
- Prokaryotes lack a nucleus, and therefore the chromosome is packed tightly into a region called the nucleoid
- Demonstrate DNA supercoiling
Regulatory sequences determine when certain genes (cell functions) are activated
Eukaryotic DNA:
- Dna wraps around histone (proteins), several come together to form nucleosome which bends and coils again and again until they become thick enough to become a chromosome held together by a centromere
- Many levels of coiling
- For most of a cells life cycle, it’s genetic material appears as a mass of long intertwined strands known as chromatin
- Eukaryotic genomes can vary a great deal between species
- Most eukaryotes are diploid (2 copies)
- Some are haploid (1 gene copy)
- Some are polyploid (more than 2 copies)
- No correlation between organism complexity and the size of its genome
- DNA identified as the hereditary material through a series of experiments
- DNA and RNA are nucleic acids composed of units called nucleotides (AGCT or U)
- Sugars in DNA deoxyribose, in RNA ribose
- Crick and Watson show DNA alpha helix
- Prokaryotic DNA arranged in nucleoid
- Eukaryotic DNA arranged in nucleus
Griffith’s experiments showed the existence of a transforming principle. That is, something in the heat-killed pathogenic bacteria (S-strain) could transform the non-pathogenic bacteria (R-strain) into a pathogenic form. This result led to Avery’s experiments on Streptococcus pneumoniae to identify the molecules that caused this transformation. Avery’s research concluded that DNA was the transforming principle.
Hershey and Chases experiment found that DNA is the hereditary material.
Miescher isolated nuclein from the nucleus of white blood cells. He found that this material was present only in the nuclei of cells. Further experimentation showed that nuclein was a weakly acidic phosphorus-containing substance. Nuclein would later be known as nucleic acid or, more specifically, DNA (deoxyribonucleic acid).
The nucleotide comp of the human be different from the nucleotide composition of the mouse because the composition of DNA is unique to each species. However, the percentage of adenine will remain approximately the same as the percentage of thymine, and the percentage of cytosine will remain approximately equal to the percentage of guanine in each species.
Levene → proposed that DNA was composed of nucleotides, and that each of the four types of nucleotides contained one of four nitrogen-containing bases, a sugar molecule, and a phosphate group.
Chargaff → showed that DNA is composed of repeating units of nucleotides in fixed proportions (i.e., the percent composition is of adenine is the same as thymine, and the percent composition of cytosine is the same as guanine). Chargaff ’s rule helped Watson and Crick infer that adenine paired with thymine, and cytosine paired with guanine.
Franklin → Franklin determined that DNA had a helical structure, with nitrogenous bases located on the inside of the structure, and the sugar-phosphate backbone located on the outside. This information led to Watson and Crick’s ladder-like double helix model of DNA, with the sugar-phosphate molecules acting as “handrails” and the bases making up the “rungs.”
Pauling → Pauling discovered that proteins have a helical structure. This discovery influenced Watson and Crick to propose that DNA was shaped like a helix.
A gene is a functional unit of DNA. It is a specific sequence that encodes for proteins or RNA molecules. A genome is an organism’s complete genetic makeup. It is composed of an organism’s total DNA sequence.
Prokaryotes Only—double-stranded, circular DNA packed in the nucleoid; DNA is compacted via supercoiling; most are haploid; genomes contain very little non-essential DNA; contain plasmids. Prokaryotes and Eukaryotes—chromosomal DNA is much larger than their cells, and therefore must be compacted. Eukaryotes Only—total amount of DNA is much greater than in prokaryotes, and they therefore have greater compacting and levels of organization (i.e., nucleosomes, chromatin, chromosomes); double- stranded linear DNA is contained in the nucleus; most are diploid; genomes can vary widely in size and complexity (i.e., some have large non-coding regions)
There is no set relationship between the complexity of an organism (number of genes in an organism) and the total size of its genome. An organism may have an enormous number of base pairs in it’s genome and very few genes if the bulk of its genome consists of non-coding DNA. Comparing the genomes of the two organisms would show what genes they have in common, and would indicate their evolutionary relationship—how closely or distantly related they are.
A mutation in a protein-coding region would not necessarily be more detrimental than a mutation in a non-coding region since the latter may contain regulatory sequences (i.e., regions that can influence the production of proteins and RNA molecules). In addition, since multiple codons exist for a given amino acid, a mutation in the protein-coding region of DNA may not alter the protein sequence.
Structure of DNA
DNA is a polymer made up of nucleotide monomers. Phosphate sugar backbone and nitrogenous base rungs → 3 h bond bw g and c but 2 with and t. 3 prime with oh, and 5 prime with oxygen. Nucleotides share a common sugar-phosphate backbone, but may possess one of 4 nitrogenous bases. G and c complementary and t and a. G and c have triple h bond (gc like groupchat, you need 3 bonds not two) and a and t have double. Rna has u instead of t
Rosalind Franklin and Wilkins:
- used x-ray diffraction analysis of DNA to determine its structure
- Franklin was able to conclude that DNA has a defined helical structure
- Franklin determined DNA has two repeating patterns at intervals of 0.34 nm and 3.4 nm
- Franklin observed how DNA reacted with water and concluded that nitrogenous bases were located on the inside of the helical structure, and the sugar-phosphate backbone was located on the outside
Watson and crick:
- Used the results, conclusions and conjectures of their peers and produced a model for DNA
- In 1962 they were awarded a Nobel Prize (along with Wilkins)
What Does DNA stand for?
Deoxyribose nucelic acid.
Describe the components and structure of a DNA molecule.
DNA molecule is made up of nucleotide monomers. Each nucleotide includes a sugar and phosphate backbone and 1 of 4 nitrogenous bases. The structure of a DNA molecule is helical.
List the 4 types of nitrogenous bases found in DNA
In DNA there is adenine, thymine, guanine and cytosine. A and T pair up while C and G pair up. A and G are purines while T and C are pyrimidines. Thre are 3 hydrogen bonds between G and C while there are only two between A and T.
Explain why DNA’s structure is called a double helix
The structure of DNA is called a double helix because each of the two strands of DNA resembles a helix in shape. A helix is a winding structure that looks like a spiral staircase. The DNA double helix is held together by hydrogen bonds that exist between the nitrogenous bases in each strand.
How are the following terms related to each other: DNA, chromatin, chromatid, chromosome?
Chromosomes are made up of two chromatids attached at the centromere and chromatids are made up of condensed chromatin. A chromatin consists of DNA and protein.
If the 6 bases on one strand of a DNA helix are AGTCGG, what are the six bases on the complementary section of the other strand?
It would be TCAGCC.
What is the difference between a histone and a nucleosome?
Histone is the protein that DNA wraps around and once it wraps around, histones form an octamer and become a nucleosome.
What is the difference between a nitrogenous base and a nucleosome?
A nucleosome is a section of DNA that is wrapped around a core of protein, while nitrogenous bases are part of the nucleotides that form DNA.
DNA replication theories
Conservative model: Two new daughter strands formed from parent templates. Original strands reform into the parent molecule
Semiconservative model: Each new molecule of DNA contains one strand of the original parent DNA and one new strand that is built complementary.
Dispersive Model: Parental DNA molecules are broken into fragments. Both strands of DNA in each daughter molecule is made of an assortment of parental and new DNA.
It was found that DNA replication uses semiconservative model. This was discovered by Meselson and Stahl.
Meselson and Stahl observed the following.
DNA samples taken just prior to the transfer to 14N media (grown in15N only) were of uniform density and appeared as a distinct band that corresponded to DNA containing only 15N, and no 14N.
After one round of replication, the DNA sample formed a single band after centrifugation. However, its position in the tube indicated that its density was midway between DNA with a nitrogen composition of only 15N and DNA with a nitrogen composition of only 14N. The researchers inferred that the DNA was composed of one strand labelled with 14N and one strand labelled with 15N. From this, Meselson and Stahl could rule out the conservative model, since it would have resulted in two bands—one band of 15N-only DNA and another band of 14N-only DNA.
After a second round of replication, the DNA sample separated into two distinct bands after centrifugation. One band corresponded to 14N-only DNA. The other band corresponded to DNA that had one strand labelled with 14N and the second strand labelled with 15N.
The same two bands that were observed after the second round of replication continued to be observed after multiple rounds of replication. This discounted the dispersive model, which would have resulted in only one band ever being observed, and supported the semi-conservative model.
DNA Replication
INITIATION: Separating a portion of the DNA double helix
ELONGATION: two new strands of DNA are made
TERMINATION: two new DNA molecules separate
Initiation:
- Starts at specific nucleotide sequences called Replication Origins
- Enzymes and other proteins work together to unwind and stabilize the double helix
- DNA Helicase: Unwinds double helix and straightens by breaking H bonds between complementary base pairs, to separate the strands
- Topoisomerase 2: Relieves the strain (from the unwinding process) on the double-helix sections ahead of the replication forks
- Single-stranded binding proteins: SSBs. Keep separated DNA strands apart by blocking hydrogen bonding
- Origion of replication is where replication starts and once the strand unwinds it results in a replication bubble
- as replication begins outwards it happens from replication forks which widen the bubble and open up the strand
Elongation:
- RECALL: DNA is double stranded and antiparallel, which means the strands run in opposite directions What defines opposite? The 5’ vs 3’ end, named for the carbon to which the phosphate group is bonded. new DNA strands are made by joining individual nucleotides together in the 5’ to 3’ direction of the new (daughter) strand
- Each strand of DNA has a 5’ (five prime) and 3’ end. DNA polymerase add complementary nucleotides from the 3’ end of the original template strand.
Leading strand:
- The daughter strand that is built continuously towards the replication fork as the double helix unwinds
- Occurs quickly
- Requires a single RNA primer (what starts the process)
- DNA polymerase 3: Catalyses the addition of new nucleotides. Only able to attach new nucleotides to the free 3’ hydroxyl end (3’OH) of the parent DNA. Attaches to the 3’ end of an RNA primer to begin process
- Primase: Builds RNA primers (used to initiate DNA replication, since polymerase can only attach to this)
Lagging strand:
- Built in short segments (in the 5’ to 3’ direction) away from the replication fork
- Requires many RNA primers
- DNA polymerase 3 adds the nucleotides
- primase starts the replication so polymerase can attach the nucleotides to it
- okazaki fragments: Short fragments of DNA built by DNA polymerase off of the RNA primers
- DNA polymerase 1: Removes the RNA primers once they have been used and replaces them with the appropriate DNA sequence
- DNA ligase: Joins the Okazaki fragments together by forming phosphodiester bonds
Termination:
- As soon as the newly formed strands are complete, they rewind automatically into their double helix
- The two new DNA molecules separate
DNA polymerase (I and II) acts as a proof-reader by checking the newly synthesized strand for any incorrectly inserted bases. If a mistake is found, act as an exonuclease – & cut out the mistake
Mismatch repair – a group of enzymes that recognize deformities
Bind to the DNA and remove the incorrect base from the daughter strand.
DNA Replication detailed info
DNA replication has to occur before cell division occurs as the daughter cells need to have the same DNA/genetic information as the parent cells.
Initiation: Replication begins at the replication origin. Helicases bind to the DNA at each replication origin. The helicases cleave and unravel a section of the original double helix, creating Y-shaped areas (replication forks) at the end of the unwound areas, which form a replication bubble. Single-strand binding proteins stabilize the separated strands. These single strands serve as templates for the semi-conservative replication of DNA.
Elongation: New DNA strands are produced when DNA polymerase inserts into the replication bubble. A primase synthesizes an RNA primer that serves as the starting point of new nucleotide attachment by DNA polymerase. DNA polymerase can only
synthesize the new nucleotide chain in the 5′ to 3′ direction. As a result, one strand (the leading strand)is replicated continuously in the 5′ to 3′ direction, in the same direction that the replication fork is moving. The other strand, known as the lagging strand, is replicated in short segments, still in the 5′ to 3′ direction, but away from the replication fork. These fragments, called Okazaki fragments, are joined together by DNA ligase.
Termination: When replication is complete, the two new DNA molecules separate from one another and the replication machine is dismantled. Each new molecule of DNA contains one parent strand and one new strand.
Early development is a very rapid process, and many key molecules are produced during this time. Therefore, it is expected that more replication origins would be present in developing embryo cells.
A replication bubble is formed as the DNA double helix unwinds during initiation. The replication forks are the Y-shaped regions of the replication bubble, and move along the DNA in opposite directions as replication proceeds.
The synthesis of a primer is necessary because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides. The primer therefore serves to prime and lay a foundation for DNA synthesis. Primers are short strands of RNA.
DNA polymerase adds new nucleotides to the 3′ end of a growing chain during replication. DNA polymerase also proofreads newly formed base pairs and replaces any nucleotides that have been incorrectly added.
a. mutation in dnaG (primase gene) → No RNA primer would be synthesized. Therefore, synthesis of the lagging strand cannot be initiated.
b. mutation in lig (ligase gene) → Okazaki fragments on the lagging strand cannot be joined together.
c. mutation in dnaB (helicase gene) → Unwinding of the DNA double helix during initiation would not occur.
- Prokaryotes Only—rate of replication is faster compared to eukaryotes; five DNA polymerases have been identified in prokaryotes; circular chromosome of prokaryotes have a single replication origin
- Prokaryotes and Eukaryotes—require replication origins; have 5′–3′ elongation; have continuous synthesis on the leading strand, and discontinuous synthesis on the lagging strand; require a primer for Okazaki fragments on the lagging strand; require the use of DNA polymerase enzymes
- Eukaryotes Only—rate of replication is slower compared to prokaryotes due to complicated enzymes complexes and proofreading mechanisms; 13 DNA polymerase enzymes have been identified in eukaryotes; linear chromosome has multiple replication origins; presence of telomeres due to linear nature of eukaryotic chromosome
Many of our tissues and organs rely on the continual regeneration of new cells, such as red blood cells and skin cells. Also, if a person is injured, new cells are needed to repair the damage. The production of new cells is achieved through mitosis and cytokinesis. Each new cell, or daughter cell, must contain the same genetic information as the original parent cell so that it can carry on the same functions. Therefore, each new cell requires an exact copy of the parental DNA. A cell copies all of its DNA only once in the cell cycle— during S phase of interphase. This process of copying one DNA molecule into two identical molecules is called DNA replication.
Watson and Crick proposed that the two strands of the DNA double-helix molecule unwind and separate, after which each nucleotide chain serves as a template for the formation of a new companion chain. The result would be a pair of daughter DNA molecules, each identical to the parent molecule. (semi-conservative model)
In the process of semi-conservative replication, each new molecule of DNA contains one strand of the original parent molecule (parent DNA) and one complementary strand that is newly synthesized (daughter DNA); each resulting DNA molecule conserves half of the original molecule. This process of replication is most often described in three basic phases that rely on the structural features of DNA and a number of specialized proteins.
- In the initiation phase, a portion of the DNA double helix is unwound to expose the bases for new base pairing.
- In the elongation phase, two new strands of DNA are assembled using the parent DNA as a template. The new DNA molecules—each composed of one strand of parent DNA and one strand of daughter DNA—re-form into double helices.
- In the termination phase, the replication process is completed and the two new DNA molecules separate from each another. At that point, the replication machine is dismantled.
Initiation:
Replication starts at a specific nucleotide sequence, called the origin of replication.
Here several initiator proteins bind to the DNA and begin the process of unwinding the double helix.
One group of proteins that is involved in this unwinding process is the helicase enzymes. The helicases cleave the hydrogen bonds that link the complementary base pairs between strands together.
Other proteins, called single-strand-binding proteins, help to stabilize the newly unwound single strands. These strands have a tendency, if unchecked, to re-form into a double helix.
These single strand regions serve as the templates that will be used to guide the synthesis of new polynucleotide strands.
In addition, the topoisomerase II enzyme helps to relieve the strain on the double-helix sections ahead of the replication forks, which results from the unwinding process.
Initiation creates an unwound, oval-shaped area called a replication bubble, with two Y-shaped regions at each end of the unwound area. Each Y-shaped area is called a replication fork.
As replication proceeds, each replication fork moves along the DNA in opposite directions.
Elongation:
The elongation phase synthesizes new DNA strands by joining individual nucleotides together.
DNA polymerase III is the enzyme that catalyses the addition of new nucleotides, one at a time, to create a strand of DNA that is complementary to a parental strand. DNA polymerase III only attaches new nucleotides to the free 3’ hydroxyl end of a pre-existing chain of nucleotides. In addition, DNA polymerase III can only synthesize a new strand from a parent strand in the 5’ to 3’ direction, toward the replication fork.
When double-stranded DNA is separated, both strands are bare templates that do not have free 3’ hydroxyl ends for DNA polymerase to begin at.
Thus, synthesis of new DNA requires both strands to be started with short fragments of nucleotide sequences complementary to the templates.
For one strand, called the leading strand, this only needs to happen once. DNA polymerase will keep adding new nucleotides to the 3’ end as it moves along in the same direction as the replication fork.
However, synthesis of the other strand requires DNA polymerase to move in the opposite direction to the replication fork.
This results in synthesis of this new strand, called the lagging strand, to occur in short segments and in a discontinuous manner. These short segments of DNA are named Okazaki fragments in honour of the scientists who identified them.
The short fragments of nucleotide sequences that are used to start or “prime” DNA replication are strands of RNA, called RNA primers. The RNA primers are synthesized by an enzyme called primase. Once a primer is in place, DNA polymerase extends the strand by adding new nucleotides to the free 3’ hydroxyl end of the primer.
For the synthesis of the lagging strand, the movement away from the replication fork necessitates several primers to be used as replication proceeds. Once each primer is added, a new DNA fragment is generated from the end of each primer.
The result is synthesis of the Okazaki fragments, a series of short fragments of DNA that each begin with a section of RNA. Eventually, another DNA polymerase enzyme, DNA polymerase I, removes the RNA primer and fills in the space by extending the neighbouring DNA fragment.
The Okazaki fragments are then joined together by the enzyme DNA ligase.
Termination:
As the replication fork progresses along the replicating DNA, only a very short region of DNA is found in a single-stranded form.
As soon as the newly formed strands are complete, they rewind automatically into their chemically stable double-helical structure.
The protein-DNA complex at each replication fork that carries out replication is often referred to as the replication machine.
The termination phase occurs upon completion of the new DNA strands, and the two new DNA molecules separate from each other.
At that point, the replication machine is dismantled.
The roles of the key enzymes in DNA replication:
- Helicase helps to unwind the parent DNA
- Primase synthesizes RNA primer used to generate Okazaki fragments
- Single-strand-binding protein helps to stabilize single-stranded regions of DNA when it unwinds
- Topoisomerase II helps to relieve the strain on the structure of the parent DNA that is generated from the unwinding of the double helix
- DNA polymerase I, II, and III a group of enzymes with differing roles that include addition of nucleotides to the 3’ end of a growing polynucleotide strand, removal of RNA primer and filling gaps between Okazaki fragments, proofreading newly synthesized DNA
- DNA ligase joins the ends of Okazaki fragments in the lagging strand synthesis
- One type of error that occurs during replication is a mispairing between a new nucleotide and a nucleotide on the template strand. For example, a T might be paired with a G instead of an A.
- Another type of error during DNA replication can be due to strand slippage, which causes either additions or omissions of nucleotides. This type of error can result from either the newly synthesizing strand looping out, allowing the addition of an extra nucleotide, or the looping out of the template strand, resulting in a nucleotide not being incorporated where it should.
One mechanism for correcting errors that occur during DNA replication involves DNA polymerases. As well as catalysing the addition of nucleotides and excising primers in DNA replication, DNA polymerase I, along with DNA polymerase II, has an important proofreading function. After each nucleotide is added to a new DNA strand, these DNA polymerases can recognize whether or not the correct nucleotide has been added.
Replication is stalled when an incorrect nucleotide is added because the 39 hydroxyl end of the incorrect nucleotide is in the wrong position for the next nucleotide to bond to it. When this occurs, these DNA polymerases excise the incorrect base from the new strand and add the correct base, using the parent strand as a template. This proofreading step repairs about 99% of the mismatch errors that occur during DNA replication.
Another mechanism for correcting DNA replication errors is called mismatch repair. This repair mechanism occurs in all species and is similar in both prokaryotes and eukaryotes. The mispairing of bases causes deformities in the newly synthesized molecule. These deformities are recognized by a group of enzymes that bind to the DNA and specifically remove the incorrect base from the daughter strand. Errors that still remain after DNA polymerase proofreading or mismatch repair are then considered mutations in the genome once cell division has occurred.
The rate of replication is faster in prokaryotes (about 1000 nucleotides added per second) than in eukaryotes (about 40 nucleotides added per second). Presumably, this is due to more elaborate enzyme complexes that are required in eukaryotic replication and a more stringent proofreading mechanism.
The DNA polymerase enzymes in eukaryotes differ from those in prokaryotes. They also differ in the number involved. To date, five have been identified in prokaryotes, while 13 have been identified in eukaryotes.
The smaller circular chromosome of a prokaryote contains a single origin of replication. The larger linear chromosome of a eukaryote may contain thousands of origins of replication.
The linear nature of eukaryotic chromosomes presents an additional problem, when the final RNA primer from the 59 end of the lagging strand is removed. For linear DNA, there is no adjacent fragment onto which nucleotides can be added and the gap filled, as there is for prokaryotes.
Therefore, when this shortened strand is copied in the next round of replication, a shorter chromosome will be produced. To ensure that this loss of DNA does not result in loss of important genetic information, the ends of eukaryotic chromosomes contain highly repetitive sequences, called telomeres. Cells have a special enzyme, called telomerase, which synthesizes these telomeric regions and can replace a sequence that has been lost.
Telomerase activity in human cells varies with development in humans. During childhood, when tissues are growing rapidly, telomerase activity is high. As people age and the rate at which tissues grow slows, telomerase activity also slows. This results in a shortening of chromosomes in somatic (body) cells. This shortening means that information from the coding portion of chromosomes may be lost.
Protein Synthesis
Transcription: making rna from dna
- diff types of rna –> all produced from a dNA template in the nucleus –> classified according to function in cell –> mRNA template for translation and tRNA and rRMA in translation of mRNA
1. Initiation: RNA polymerase binds to promoter region (sequence of DNA upstream of the gene to be transcribed) – opens up the double helix –> promoters contain base pair patterns rich in As and Ts →Called a TATA box –> RNA polymerase binds to DNA at promoter region –> DNA strand is unwound, double helix disrupted…exposing template strand
2. elongation: RNA polymerase starts building mRNA in the 5’ to 3’ direction. process similar to DNA replication – except… no primer is used. only 1 strand of DNA is used -the template strand or the antisense strand. DNA template is used to synthesize mRNA in 5’ to 3’
(uracil complements adenine). DNA that has already been transcribed rewinds into double helical form. unused DNA strand is called the coding strand or the sense strand. multiple mRNA strands are synthesized simultaneously from one DNA template →ie. many bubbles. ***mRNA is complementary to the template strand and identical to the coding strand (except it contains U)
3. Termination: Specific NT sequences in the template serve as a signal to stop transcription. mRNA dissociates from the DNA template. RNA polymerase is free to transcribe another gene. DNA dbl helix reforms
Post transcription modifications: mRNA MODIFICATIONS in Eukaryotes
- Convert precursor mRNA (pre-mRNA) to mature mRNA
- 1) 5’ cap added. 7-methyl guanosine added to 5’ end. Cap is recognized by the protein synthesis machinery
2) Poly-A tail added. The addition of a series of A nucleotides to the 3’ end of the pre-mRNA. Makes mRNA more stable, allows it to exist longer in cytoplasm
3) Introns cut out. eukaryotic genes have coding regions called exons, and non-coding regions called introns. Splicing – intron sequences are removed and the exons are joined together to form the mature mRNA. mRNA leaves the nucleus following these modifications
Unlike DNA replication, no quality control in mature RNA, therefore, there are more errors made during transcription than DNA replication….BUT…
Since a single gene is transcribed many times to produce 100’s of transcripts, errors are not as detrimental.
Errors in mRNA result in a protein being made that is susceptible to degradation
Translation: synthesizing proteins from mRNA
Machinery:
mRNA: Genetic information that determines the amino acid sequence in a protein
tRNA: Anticodon with amino acid attached that base-pairs with mRNA codon (reads rna, complementary to rna)
Ribosomes: Composed of rRNA and proteins (machine that allows mrna to attach to while trna does it’s job)
Translation factors: Accessory proteins used during translation
tRNA: Transfer RNA:
clover-leaf shaped RNA with three double-stranded stem-loops held by intramolecular base pairing, and one
3’ single-stranded region
Two functional regions:
Anticodon loop: three nucleotides; complementary to an mRNA codon (recall: 3 nucleotide amino acid code)
Acceptor stem: 3’ single-stranded end directly across from anticodon loop; site of amino acid attachment via aminoacyl-tRNA synthetase (20 = 1 per amino acid)
Genetic code has 64 possible combinations
Ribosomes:
Proteins + ribosomal RNA (rRNA)
Serve as the “hub” where mRNA, tRNA, and enzymes work together to carry out protein synthesis
Has one binding site that attaches to mRNA, and three binding sites for tRNA attachment (amino acid site = A, peptide site = P, exit site = E)
RECALL: during transcription, multiple mRNA strands are synthesized simultaneously from one DNA template
Similarly, multiple ribosomes can simultaneously translate a single mRNA strand → called a polyribosome
Initiation: Initiation factor proteins assemble ribosomes, mRNA, intiator tRNA at P site on ribosome with anticodon UAC (and methionine) at the start codon AUG on mRNA.
Elongation and termination:
- A second, incoming tRNA with amino acid enters A-site.
- Initiator tRNA at P-site transfers methionine to amino acid at A-site, forming a peptide bond. The developing polypeptide is now at the A-site, momentarily.
- As the ribosome moves along the mRNA to the next codon, the polypeptide-bearing tRNA shifts from A-site to P-site, and the initiator tRNA shifts from P-site to E-site, where it then exits the ribosome.
- Another new amino acid carrying tRNA enters the now empty A-site and the process repeats until termination.
- Termination occurs when the ribosome arrives at a stop codon on the mRNA. The polypeptide is released, folds into its 3D shape, and the machinery disassembles.
Transcription
DNA is often described as the blueprint of a cell, which stores information needed for survival and reproduction.
The first step in how this genetic information is read involves the production of an RNA molecule.
Like DNA, RNA is a polymer of nucleotides. Recall that RNA contains four nucleotides with the bases adenine (A), uracil (U), cytosine (C), and guanine (G). Unlike DNA, RNA is single-stranded. However, it can fold back on itself, and complementary base pairing within the same molecule stabilizes the looped structure. Have ribose sugar. Can move in and out of the nucleus.
There are several types of RNA molecules.
Since all are produced from a DNA template, all are synthesized in the nucleus. However, they are classified according to the different functions they have in the cell.
For example, mRNA acts as an intermediary between the DNA sequence of a gene and protein synthesis. The mRNA is used as a template that determines the amino acid sequence of the protein that it codes for. There are many RNAs that do not code for proteins, but that still have essential functions in the cell. Two of these are involved in the process of translation: tRNA and rRNA
The Molecular Events of Transcription:
The main objective for transcription is to accurately produce a copy of a small section of genomic DNA.
In a similar manner to what occurs in DNA replication, there are three defined stages in the transcription process: initiation, elongation, and termination.
Note that transcription in eukaryotes and prokaryotes is very similar. The main differences are the proteins involved. More proteins are required for each phase of transcription in eukaryotes, and they form more complex associations.
Initiation:
During the initiation phase, the correct position for transcription to start is selected and the transcription machinery, composed of a large protein-DNA complex, is assembled.
For each gene, only one strand of the double-stranded DNA molecule is transcribed. This strand is called the antisense strand or template strand.
The other strand, which is not transcribed, is called the sense strand or coding strand. It has the same sequence as the product mRNA, with thymines instead of uracils.
In a single DNA molecule, either strand can serve as the sense strand for different genes.
The main enzymes that catalyze the synthesis of RNA are a group of enzymes called RNA polymerases. In eukaryotes, each RNA polymerase has a specific function. Once the RNA polymerase complex has bound to the DNA molecule, it unwinds and opens a section of the double helix.
Transcription begins when RNA polymerase binds tightly to a promoter region on the DNA. This region contains special sequences of nucleotides.
Notice how there are two sets of sequences. Both of these are required and need to be correctly positioned relative to each other. This allows the RNA polymerase complex to bind to the correct strand and in the correct orientation, so that the strand is copied in the correct direction.
Elongation:
During the elongation phase the RNA polymerase complex works its way along the DNA molecule, synthesizing a strand of mRNA that is complementary to the template strand of DNA.
In the mRNA strand, however, T is replaced with U.
Like DNA polymerase, RNA polymerases work in the 59 to 39 direction, adding each new nucleotide to the 39–OH group of the previous nucleotide.
RNA polymerases transcribe only one strand of the template DNA, so there is no need for Okazaki fragments, as in DNA replication.
As soon as the RNA polymerase complex starts to move along the DNA, a second RNA polymerase complex can bind to the promoter region and start to synthesize another mRNA molecule.
this means that hundreds of copies of mRNA molecules can be made from one gene at one time.
Also, RNA polymerase catalyzes the synthesis of mRNA at a much faster rate than DNA polymerase catalyzes the synthesis of DNA. This is mainly because the RNA polymerase complex does not have a proofreading function.
An error in transcription would only result in an error in one protein molecule, and not in the genetic make-up of an organism. Being able to synthesize more mRNA in a given amount of time is a greater advantage than minimizing sequence errors.
Termination:
For the termination phase, specific nucleotide sequences in the template DNA serve as a signal to stop transcription.
When the RNA polymerase complexes reach this signal, they detach from the DNA strand.
The new mRNA strand is released from the transcription assembly, and the DNA double helix reforms.
mRNA modification in eukaryotes:
In prokaryotes, an mRNA molecule can be used in protein synthesis as soon as it is made. In fact, transcription and translation can occur simultaneously.
In eukaryotes, the newly synthesized mRNA undergoes modifications before it is transported across the nuclear membrane into the cytoplasm.
These modifications convert precursor mRNA (pre-mRNA) to mature mRNA
- Addition of a 59 cap. This involves the covalent linkage of a modified G nucleotide to the 59 end of the pre-mRNA. The cap is recognized by the protein synthesis machinery.
- Addition of a 39 poly-A tail. This involves the covalent linkage of a series of A nucleotides to the 39 end of the pre-mRNA. The tail makes the mRNA more stable and allows it to exist longer in the cytoplasm.
- Removal of introns. Eukaryotic genes contain non-coding regions called introns, which are interspersed among coding regions called exons. The intron sequences are removed from pre-mRNA, and the exons are joined together to form the mature mRNA. This process, called splicing, is shown in Figure 6.10. Particles composed of snRNA and proteins, called snRNPs (pronounced “snurps”), recognize regions where exons and introns meet, and they bind to those areas. The snRNPs interact with other proteins, forming a larger spliceosome complex that removes the introns. For expression of most genes, all of the exons are spliced together. In some cases, however, only certain exons are used to form a mature RNA transcript. This alternative splicing, therefore, allows for one gene to code for more than one protein. As a result, certain cell types are able to produce forms of a protein that are specific for that cell.
Translation
The second stage of gene expression involves translating the nucleic acid code of mRNA into the amino acid code of a protein.
This process requires the assembly of a complex translation machinery composed of different nucleic acid and protein components.
mRNA contains genetic information that determines the amino acid sequence of a protein
tRNA contains an anticodon that base-pairs with a codon on the mRNA and has the corresponding amino acid attached to it, according to the genetic code
Ribosomes composed of rRNA and proteins; involved in the process of protein synthesis
Translation factors proteins that act as accessory factors; needed at each stage of translation
Transfer RNA (tRNA) molecules are composed of a single strand of RNA that folds into the characteristic two-dimensional cloverleaf shape.
tRNA consists of three stem-loops and a single-stranded region, which in turn fold into a three-dimensional boot-shaped structure.
The stem-loops are areas of double-stranded RNA that form through intramolecular base pairing.
Each tRNA molecule has two functional regions. One contains the anticodon loop, which is a sequence of three nucleotides that is complementary to a specific mRNA codon.
At the opposite end of the molecule, at the 39 single-stranded region, is the acceptor stem, where an amino acid is attached.
The aminoacyl-tRNA synthetase enzymes are responsible for attaching the appropriate amino acid to a tRNA according to its anticodon.
There are 20 different enzymes, one for each of the 20 amino acids. This reaction must be very precise, because tRNAs are responsible for delivering the amino acids to the translation machinery.
The correct amino acid must be linked to the appropriate mRNA codon.
Recall that when nucleotide sequences are given, they are written in the 59 to 39 direction. Anticodons are written in the 39 to 59 direction.
Ribosomes are made up of proteins as well as ribosomal RNAs (rRNAs) that are bound to the proteins.
They are cytoplasmic structures that provide a place where the mRNA, tRNAs that carry the amino acid molecules, and enzymes involved in protein synthesis can assemble and interact.
each ribosome is comprised of two sub-units. Each sub-unit is composed of different proteins and rRNA molecules.
The ribosome has a binding site for the mRNA and three binding sites for tRNA molecules. These binding sites permit complementary base-pairing between tRNA anticodons and mRNA codons. As soon as the initial portion of mRNA has been translated by one ribosome and the ribosome has begun to move down the mRNA, another ribosome attaches to the same mRNA. Therefore, several ribosomes are often attached to and translating a single mRNA.
This whole complex is called a polyribosome, and it can synthesize several copies of a polypeptide at the same time.
Translation is one of the most energy-consuming processes of the cell. Many protein and nucleic acid components must be synthesized and assembled through a complex array of steps, and energy is needed each time two amino acids are bonded together.
Translation involves an mRNA being threaded through a ribosome, with the codons of the mRNA base-pairing with the anticodons of tRNA molecules that carry specific amino acids.
The order of the codons determines the order of the tRNA molecules at the ribosome and, thus, the sequence of amino acids in a polypeptide.
Translation can be divided into three phases: initiation, elongation, and termination.
Initiation:
In the initiation phase, all the translation components come together.
Proteins called initiation factors assemble the small ribosomal sub-unit, mRNA, initiator tRNA, and the large ribosomal sub-unit for the start of protein synthesis.
the small ribosomal sub-unit attaches to the mRNA near the start codon (AUG).
The first tRNA that binds to the codon is the initiator tRNA with its UAC anticodon. In prokaryotes, this tRNA carries a derivative of methionine. In eukaryotes, methionine is used.
Then, a large ribosomal sub-unit joins to form the active ribosome.
The start codon sets the reading frame for the gene. The reading frame establishes how all subsequent codons in the sequence will be read.
The three binding sites for tRNAs are the P (peptide) site, the A (amino acid) site, and the E (exit) site.
During protein synthesis, the P site contains the tRNA with the growing polypeptide attached to it; the A site contains the tRNA with the next amino acid to be added to the polypeptide chain; and the uncharged tRNA, which no longer has an amino acid attached to it, is ejected at the E site.
At initiation, the initiator tRNA binds to the P site.
Elongation:
In the elongation phase, protein synthesis occurs. The polypeptide becomes longer, one amino acid at a time.
In addition to tRNAs, elongation requires elongation factors, which enable tRNA anticodons to bind to mRNA codons.
Once the initiator tRNA is bound to the ribosome, the A site is occupied by the tRNA with an anticodon that base-pairs with the second mRNA codon, and that carries the second amino acid of the protein being synthesized. A peptide bond forms between the first and second amino acids, and the resulting dipeptide is attached to the tRNA at the A site.
The mRNA moves along by one codon, and this complex becomes associated with the P site of the ribosome. The sequential addition of amino acids during elongation is a cycle of four steps that is rapidly repeated.
First, a tRNA with an attached polypeptide is in the P site and tRNA carrying the next amino acid enters the A site. Next, the polypeptide chain is transferred to the amino acid of the tRNA in the A site. This makes the polypeptide chain one amino acid longer than before. Last, the mRNA moves forward by one codon, and the polypeptide-bearing tRNA is now at the ribosome P site. The uncharged tRNA exits. The new codon is at the A site and can receive the next complementary tRNA carrying the next amino acid of the polypeptide.
Termination:
The termination phase begins when a stop codon on the mRNA is reached.
The polypeptide and the components of the translation machinery are separated.
A protein, called a release factor, cleaves (cuts) the polypeptide from the last tRNA. The polypeptide is released and will eventually fold into its three-dimensional shape, ready to carry out its cellular activities.
Gene expression and mutation
The expression of a gene refers to the synthesis of a protein that is encoded by that gene.
Protein synthesis requires the processes of transcription and translation.
During transcription, the DNA sequence of a gene serves as a template for the synthesis of mRNA. In eukaryotes, the mRNA is processed before it leaves the nucleus, which involves removal of introns and addition of a 59 cap and a 39 poly-A tail.
During translation, the mRNA carries the genetic information to the ribosomes, where protein synthesis occurs. The translation machinery includes tRNAs, which bring amino acids to the ribosomes and act as carriers of the growing polypeptide chain.
The underlying mechanisms for how the DNA sequence information of a gene is decoded to an amino acid sequence of a protein is summarized in Figure 6.16.
During transcription, the sequence in a region of DNA is copied into a sequence of mRNA codons. Translation of the mRNA codons to an amino acid sequence occurs through tRNA molecules. The amino acid-charged tRNA molecules have anticodons that base-pair with the codons. The order of the codons of the mRNA determines the order that the tRNA-amino acids bind to a ribosome. This determines the order that the amino acids are incorporated into the growing polypeptide chain that will become the complete protein.
DNA Mutations and Effects of Mutagens:
In the dynamic environment of a cell, DNA is constantly being replicated, and errors do occur.
Enzymes quickly repair some errors. Others can result in a mutation—a change in the genetic material of an organism.
Because the DNA is changed, all mutations are copied during DNA replication and passed to daughter cells.
As well, mutations that occur in reproductive cells can be passed on from one generation to another. Mutations in somatic (body) cells, on the other hand, do not affect future generations.
Mutations are typically neutral or harmful to an organism. In rarer cases, they may be beneficial. Beneficial mutations that affect future generations are important in terms of species change and adaptation over time.
Mutations fall into one of two general categories, single-gene mutations and chromosome mutations.
Single-gene mutations involve changes in the nucleotide sequence of one gene.
Conversely, chromosome mutations involve changes in chromosomes, and may involve many genes.
A single-gene mutation resulting from a change in a single base pair within a DNA sequence is a point mutation.
A point mutation can involve the substitution of one nucleotide for another. It can also involve the insertion or deletion of a single base pair.
A point mutation that involves a nucleotide substitution may have a fairly minor effect on the cell. One reason for this is the redundancy of the genetic code. A change in the coding sequence of a gene does not always result in a change to the polypeptide product of the gene.
While nucleotide substitutions do not affect neighbouring coding sequences, the insertion or deletion of a number of nucleotides that is not divisible by three can do so. Such an insertion or deletion results in a frameshift mutation. A frameshift mutation causes the entire reading frame of the gene to be altered
Mutations also may be categorized by how they affect the amino acid sequence of a protein.
A mutation that has no effect on the amino acid sequence of a protein is called a silent mutation.
A mutation that does result in an altered amino acid sequence of a protein is called a missense mutation. Missense mutations can be harmful.
The most common mutation results in one amino acid change in the enzyme, which affects the activity of the enzyme.
Missense mutations can also develop new proteins that can help an organism survive.
On the other hand, if a change in a gene’s coding sequence results in a premature stop codon, then a shortened protein or no protein at all will be made. Such a mutation is called a nonsense mutation. These mutations are usually harmful to an organism.
changes to the chromosome number in an organism’s genome are always detrimental and often lethal.
Mutations can also involve a rearrangement of genetic material, which may affect several genes, including genes located on different chromosomes.
Changes to chromosome structure include the deletion or duplication of portions of chromosomes. There can also be inversions, in which a segment of a chromosome is broken and then re-inserted in the opposite direction. Finally, translocations involve a section of one chromosome breaking and fusing to another chromosome.
Incorrect base pairing by DNA polymerase during DNA replication is one source of spontaneous mutations.
Another cellular process, called DNA transposition, can disrupt more extensive regions of genetic information. DNA transposition involves the movement of specific DNA sequences within and between chromosomes. Because these sequences effectively swap or exchange places, they are referred to as transposable elements, or transposons for short.
All cells can undergo spontaneous mutation, but exposure to certain factors in the environment can increase the rate of mutation. Mutations that are caused by agents outside the cell are referred to as induced. A substance or event that increases the rate of mutation in an organism is called a mutagen. Mutagens may be physical and chemical.
x-rays are physcial mutagens (causes change in the dna)
A chemical mutagen is a molecule that can enter the nucleus of a cell and induce mutations by reacting chemically with the DNA.
A chemical mutagen may cause a nucleotide substitution or a frameshift mutation. Other chemical mutagens have a structure similar to that of ordinary nucleotides but with different base-pairing properties.
When these mutagens are incorporated into a DNA strand, they can cause incorrect nucleotides to be inserted during DNA replication.
Examples of chemical mutagens include nitrites (found in small amounts in cured meats), gasoline fumes, and more than 50 different compounds in cigarette smoke.
Most chemical mutagens are carcinogenic (associated with one or more forms of cancer).
Mutations and the phenotypic variations that can result from them may have a positive, neutral, or negative effect on an organism. If the effect is positive, the variation may be passed on to an increasing number of organisms in a population and may lead to the evolution of an existing species or the development of a new species.
However, mutations that accumulate too rapidly or are very harmful do not provide a selective advantage.
Therefore, cells have a variety of DNA repair mechanisms.
Cells have many other mechanisms that can repair DNA. These all involve a specific set of proteins that act by recognizing and then repairing the damage. These mechanisms are either specific or non-specific.
Specific repair mechanisms fix certain types of damage.
repair is non-specific because it can correct different forms of damage.
Transfer of Info from DNA
Establishing a Link between Genes and Protein:
The idea of a link between genes and proteins was first introduced by an English chemist and physician, Archibald Garrod (1857–1936).
In 1902, Garrod published his studies of patients with a disease called alcaptonuria. The disease causes urine to turn black when it is exposed to air. The colour change is caused by increased levels of homogentisic acid in urine.
Garrod’s approach was to investigate the disease biochemically, as a series of chemical reactions.
He correctly proposed that the build-up of homogentisic acid was due to a defective enzyme in the metabolic pathway that breaks down the amino acid phenylalanine.
When Garrod discovered that certain patients were blood relatives, he hypothesized that alcaptonuria was an inherited disease.
Garrod suggested that the black urine phenotype was due to what Mendel called a recessive inheritance factor. Having this defective factor resulted in the production of a defective enzyme.
From their studies, Beadle and Tatum concluded that one gene specifies one enzyme.
They called this relationship the one-gene/one-enzyme hypothesis. In 1958, Beadle and Tatum shared half of the Nobel Prize in physiology or medicine “for their discovery that genes act by regulating definite chemical events.”
The original hypothesis was later updated to account for the fact that not all proteins are enzymes, and that many enzymes are composed of more than one polypeptide chain. Thus, it is now more commonly called the one-gene/one-polypeptide hypothesis.
In 1953, the English biochemist Frederick Sanger showed that proteins consist of amino acids covalently linked together
. Studying the protein insulin, Sanger established that each insulin molecule was made up of the same sequence of amino acids. Therefore, each protein has a particular amino acid sequence. This discovery came at the same time that Watson and Crick determined that DNA consists of strands of nucleotide sequences.
By the early 1960s, a clear link between genes and proteins also had been made, but the nature of this link was still a mystery.
In eukaryotes, genes were known to be located on chromosomes that occur only in the nucleus. As well, protein synthesis in eukaryotes was known to occur only in the cytoplasm. Thus, scientists could discount the idea that proteins were directly synthesized from DNA.
Some evidence supported the idea that another type of nucleic acid, RNA, was an intermediary between DNA and proteins. This evidence included the fact that RNA could be found in both the nucleus and cytoplasm of eukaryotes. As well, the concentration of RNA in the cytoplasm correlated with the level of protein production. Researchers also studied where RNA is synthesized in the cell, and they followed its transport within the cell.
Through these studies, they were able to show that RNA is synthesized in the nucleus and then transported to the cytoplasm.
In 1961, Francois Jacob and Jacques Monod hypothesized the existence of a special type of RNA that acts as a “genetic messenger.” They proposed that this RNA, which they called messenger RNA (mRNA), is synthesized from the DNA of genes. The mRNA base sequence would be complementary to the gene DNA sequence. This mRNA nucleotide sequence would provide the amino acid sequence information needed for protein synthesis.
In 1964, Jacob, working with Sydney Brenner and Mathew Meselson, confirmed the messenger RNA hypothesis. The results of their experiment showed the following:
When bacteria were infected by a virus, a virus-specific RNA molecule was synthesized and became associated with pre-existing bacterial ribosomes. Recall that ribosomes are the site of protein assembly in the cell.
The new RNA molecule had a base sequence complimentary to the DNA and carried the genetic information to produce the viral protein.
This viral RNA molecule was newly synthesized and was not a permanent part of the bacterial ribosomes.
Once scientists established that mRNA acts as an intermediary, they turned their attention to “cracking” the genetic code.
This code would help them understand how information is converted from the nucleotide sequence of an mRNA to the amino acid sequence of a protein.
Researchers knew that are were only four nucleotides in RNA (A, U, G, and C), but 20 different amino acids. Therefore, there could not be a one-to-one relationship between nucleotide and amino acid.
Even using two nucleotides per amino acid would only provide 4 × 4, or 16 possible combinations, which is not enough to code for 20 amino acids.
Thus, the minimum combination of the four nucleotides was a triplet code, which could produce 4 × 4 × 4, or 64 possible combinations.
From this reasoning came the triplet hypothesis, which proposed that the genetic code consists of a combination of three nucleotides, called a codon.
The most convincing support of the triplet hypothesis came from the 1961 studies by Francis Crick and South African biologist Sydney Brenner. These researchers used T4 bacteriophages, which are viruses that infect E. coli. Nucleotides were either added to or deleted from the DNA sequence.
Crick and Brenner found that when they added or deleted one nucleotide or a pair of nucleotides, the viral protein was not produced. This result could be reversed if additional mutations were able to reinstate the triplet codon. For example, if a nucleotide had been deleted, the addition of a nucleotide resulted in the production of the viral protein and E. coli infection. Also, if the mutation added or deleted three nucleotides that were close together, viral protein production and bacterial infection occurred. Crick and Brenner showed that the genetic code it is read in triplets, with no spaces in the code—it is read continuously.
Now that the idea of a genetic code based on multiples of three nucleotides was established, the race was on to determine which codons specified specific amino acids.
Between 1961 and 1965, various research groups compared artificially synthesized RNA molecules of known nucleotide sequences with the amino acid sequences of polypeptides.
From these studies, the mRNA codons and their corresponding amino acids were determined.
By convention, the genetic code is always interpreted in terms of the mRNA codon rather than the nucleotide sequence of the DNA.
The genetic code has three important characteristics.
1. The genetic code is redundant. This means that more than one codon can code for the same amino acid. There are only three codons that do not code for any amino acid. As you will learn later in the chapter, these codons serve as “stop” signals to end protein synthesis.
2. The genetic code is continuous. This means that it reads as a series of three-letter codons without spaces, punctuation, or overlap. Therefore, knowing exactly where to start and stop protein synthesis is essential. A shift of one or two nucleotides in either direction can alter the codon groupings and result in an incorrect amino acid sequence.
3. The genetic code is nearly universal. Almost all organisms build proteins with the genetic code shown in Table 6.1. (Some rare exceptions are known in some species of protists, for example.) The universality of the genetic code means that a codon in the fruit fly codes for the same amino acid as in a human. this has important implications for genetic techniques, such as cloning. A gene that is taken from one kind of organism and inserted into another kind of organism will produce the same protein.
Gene expression refers to the synthesis of a protein based on the DNA sequence of a gene.
the path of gene expression. The theory that genetic information flows from DNA to RNA to protein is often called the central dogma of genetics.
The two steps in the expression of a gene are transcription and translation.
In transcription, mRNA is synthesized based on the DNA template of a gene.
This is followed by translation, which involves the production of a protein with an amino acid sequence that is based on the nucleic acid sequence of the mRNA. The translation of nucleotide sequence to amino acid sequence uses the genetic code.
Regulating Gene Expression in Prokaryotes and Eukaryotes
Gene Regulation:
Control of the level of gene expression, gene activity/inactivity in response to cellular/environmental demands
Most genes require some level of regulation
Constitutive genes: always active, constantly expressed gene whose proteins are essential to survival (aka housekeeping genes)
In prokaryotes it is regulated during three different levels:
Transcription, Translation and After protein synthesis
Recall: RNA polymerase complex binds at the promoter region to begin transcription
In bacteria, promoters control clusters of genes called operons
Genes involved in the same metabolic pathway are grouped together in the same operon, and are transcribed together into a continuous, polycistronic mRNA strand
lac Operon:
Consists of genes that code for enzymes needed to break down lactose sugar in E. coli when lactose is the available food source
Coding region: contains genes for three enzymes required for lactose metabolism
- Regulatory region contains:
promoter for transcription of the enzymes
operator 🡪 site that regulates transcription via inhibition by binding to a repressor protein in the absence of lactose
CAP binding site 🡪 DNA sequence to which the activator protein, CAP, binds to increase transcription rates in the presence of lactose
trp Operon:
Coding region contains five genes for enzymes required for tryptophan synthesis
Usually transcribed until the cell has sufficient tryptophan, at which point the trp repressor binds to the promoter to inhibit transcription
Gene Expresion in Eukaryotes:
Does not have operons
Five levels of regulation:
1. Pre-transcriptional 🡪 transcription factors initiate transcription to a certain level; activators enhance initiation by binding to transcription factors and RNA polymerase
2. Transcriptional 🡪 multiple activators to maintain regulation
3. Post-transcriptional 🡪 occurs at the mRNA level (made enough mrna)
4. Translational 🡪 RNA interference inhibits gene expression by degrading mRNA or inhibiting translation (message destroyed)
5. Post-translational 🡪 once polypeptide is formed, protein activation requires modifications of the chain; other specialized molecules degrade proteins when they are no longer needed (if we didn’t stop soon enough)
Regulation of Genes detailed
Gene regulation refers to control of the level of gene expression. This regulation not only involves whether a gene is active or inactive, but also determines the level of activity and amount of protein that is available in the cell.
Some genes are always active and expressed essentially at constant levels, because the proteins are needed for survival of the cell. These are called constitutive genes, or housekeeping genes.
Most genes, however, are regulated so that the protein is expressed only at certain times and in certain amounts.
In Prokaryotes:
Regulation of gene expression in prokaryotes can occur at three different levels: during transcription, during translation, and after the protein is synthesized.
By far the most common form of regulation is at the level of transcription, during initiation.
In bacteria, many genes are clustered together in a region that is under the control of a single promoter. (Recall that the promoter is where the RNA polymerase complex binds to DNA and begins transcription.) These regions are called operons. Genes that are involved in the same metabolic pathway are often found in the same operon.
Since they are all under the control of the same promoter region, these genes are all transcribed together into one continuous mRNA strand, called a polycistronic mRNA. Individual proteins are then synthesized from that mRNA.
lac operon:
E. coli can use different sugars as a source of energy and carbon. It does this by quickly adjusting its gene expression according to what sugar is available.
For example, when the bacteria are grown in the presence of the sugar lactose, there is an increase in the levels of enzymes that are involved in lactose metabolism.
When the lactose is subsequently removed, the levels of these enzymes drastically decline.
The genes that encode the enzymes that are needed to break down lactose are found in the lac operon on the E. coli chromosome.
This operon consists of a coding region and a regulatory region.
The coding region contains the genes for three enzymes that are required for the breakdown of lactose.
The regulatory region contains the promoter for transcription of the lactose-metabolizing enzymes. It also contains sites that regulate that transcription, an operator, and a catabolite activator protein (CAP) binding site.
An operator is a DNA sequence to which a protein binds to inhibit transcription initiation. This protein is called a repressor.
The CAP binding site is a DNA sequence to which a specific protein also binds. The binding of CAP increases the rate of transcription of a gene or genes.
In the absence of lactose, the lac repressor protein binds to the operator. This prevents RNA polymerase from binding to the promoter, and transcription cannot occur.
When lactose is present, a derivative called allolactose is produced. Allolactose binds to the repressor and the repressor can no longer bind to the operator. This results in the transcription of the genes to produce the required enzymes.
The binding of the CAP activator protein can even further enhance levels of transcription.
The lac operon is considered an inducible operon. Transcription from it is induced when lactose is present. There are also other operons that are normally active until a repressor turns them off. An example of this type of operon is the trp operon.
trp operon:
The coding region contains five genes for enzymes that are required for the synthesis of the amino acid tryptophan.
The regulatory region contains a promoter and an operator region.
Under normal conditions, tryptophan must be synthesized, so the repressor does not bind to the operator, and transcription takes place.
When tryptophan reaches a certain level in the cell, however, some of it binds to a repressor protein. This binding increases the repressor’s ability to bind to the operator, thus reducing transcription activity.
In eukaryotes:
There are five levels of gene regulation in eukaryotes: pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational.
PreTranscriptonal and Transcriptional Control:
Regulation of gene expression can take place at the DNA level.
Recall that DNA is associated with proteins such as histones to form nucleosomes. These, in turn, assemble into more condensed structures to form chromatin.
DNA in highly condensed areas of chromatin is not transcribed, because chromatin acts as a physical barrier to the proteins that are needed to synthesize pre-mRNA.
For regions of the genome that need to be expressed, different processes are used by the cell to alter chromatin structure and loosen the nucleosome structures. Once this is done, proteins for initiation of transcription can gain access to the DNA.
Methods for accomplishing alterations include chemical modifications of the histone proteins and the use of multi-enzyme structures called chromatin-remodelling complexes.
Eukaryotic genes are not organized into operons. Rather, each gene has its own promoter, and control of transcription is distinct for that gene.
Transcription factors must interact with the promoter for RNA polymerase to initiate transcription.
Although this interaction is essential, it only allows a certain level of transcription. Certain types of activator proteins also enhance transcription initiation by binding to transcription factors and RNA polymerase, as well as specific sequences of DNA called enhancers.
There are a vast array and number of activators in eukaryotes, and regulation of a gene usually requires more than one type. Having multiple activators allows gene regulation to be highly tuned to particular conditions or times during the life of the cell.
Post Transcriptional and Translational Control:
Regulation of gene expression can also occur at the level of the mRNA molecule, once it is synthesized.
As discussed earlier, alternative splicing of an mRNA produces different mRNA molecules. These, in turn, provide alternative protein products.
Modifications of the mRNA can be altered so that the 59 cap and/or 39 poly-A tail are not added. mRNAs lacking in the modification will either not be transported from the nucleus or undergo rapid degradation in the cell. In either case, the mRNA is no longer available for protein synthesis in the cytoplasm.
Small RNA molecules can control gene expression by a mechanism called RNA interference. Two of these small RNAs are micro RNA (miRNA) and small interfering RNA (siRNA). These small RNAs can associate with protein complexes and turn off gene expression by either promoting mRNA cleavage or inhibiting translation. The small RNAs can target and interact with specific mRNAs by forming complementary base pairs.
Post Translational Control:
Many polypeptides that are synthesized in eukaryotic cells are not active immediately after synthesis.
This activation involves one of a number of different modifications. For example, insulin is initially folded into its three-dimensional structure. However, in order to be active several amino acids are removed, which leaves two polypeptide chains. The chains are combined by the formation of a covalent bond between two sulfur atoms that are on each chain. This activates the insulin protein.
Other modifications include the covalent linkage of a phosphate group to one or more amino acids in the polypeptide in order for the protein to be functional.
Regulating how long a protein is available in the cell can also be a form of gene regulation
An important pathway that eukaryotic cells have for this involves the attachment of a chain of ubiquitin molecules to a protein. This acts as a signal for the protein to be degraded.
Extra gene regulation info
Housekeeping genes regulate important processes such as metabolism, growth, and DNA replication and transcription. They are continuously transcribed and translated because these proteins are always needed in the cells for basic cellular function. (In molecular biology, housekeeping genes are typically constitutive genes that are required for the maintenance of basic cellular function, and are expressed in all cells of an organism under normal and patho-physiological conditions.)
lac Operon:
Lactose is an energy source for prokaryotes - obtained directly from the environment.
To regulate the expression of the genes that are required for lactose metabolism, prokaryotes use what is known as the lac operon model of gene expression.
The lac operon is an example of an inducible set of genes.
These genes are responsible for the breakdown of lactose into sugars used for cellular metabolism.
Operon is a cluster of linked genes that function together and regulated by one promoter.
The lac Operon consists of a promoter, an operator and the coding region for the various enzymes that actually metabolizes the lactose.
A promoter is the site where DNA transcription begins.
An operator is the sequence of bases that control transcription.
Repressor protein - protein that binds to the operator to repress gene transcription - regulates the production of lactose-metabolizing proteins.
For the lac operon, the repressor protein is called the lacI protein or lac repressor.
When lactose is absent, from the cell’s environment, the active lac repressor protein blocks transcription of lac operon genes by binding to the lac operator and partially blocking RNA polymerase.
This prevents RNA polymerase from reaching and transcribing the genes further down the strand.
When lactose is present in the cell’s environment, the lac repressor must be removed so transcription takes place.
Lactose acts as a signal molecule (inducer) by binding to lac repressor protein.
lac repressor changes its configuration and can no longer bind to operator - this allows RNA polymerase to bind to promoter, and transcription of the lac operon genes.
Translation of the mRNA produces the three lactose metabolism enzymes
trp Operon:
Tryptophan is an important amino acid that is used by bacteria to build protein.
If it is not found in its environment, the bacteria will make its own.
The operon that regulates the production of tryptophan is called the trp operon.
The trp operon has the same structure as the lac operon: a promoter and an operator that precedes the 5 genes coding for tryptophan-synthesizing enzymes.
There is also a gene that codes for a trp repressor protein - activated in the presence of tryptophan
When tryptophan is absent from the environment, the trp repressor is inactive in binding to the operator.
Thus, the RNA polymerase can transcribe the genes that ultimately produce tryptophan.
When tryptophan is present in the environment, tryptophan binds to the trp repressor protein altering its shape - now active.
The trp repressor-tryptophan complex binds to the trp operator.
RNA polymerase is unable to bind to promoter region and the genes are not expressed.
Since tryptophan is needed to inactivate the trp operon, it is called a corepressor.
A cell responds to change in its environment by regulating the rate at which genes are expressed.
Prokaryotes use operons to regulate gene expression.
An operon is a section of DNA that includes a promoter, an operator, a regulatory protein and the sequence of genes that code for one or more specific protein.
The lac operon uses a signal molecule (lactose) that induces the expression of the operon genes
The trp operon uses a signal molecule (tryptophan) that represses the expression of the operon genes.
Gene regulation in eukaryotes can occur during transcription, post-transcription, translation and post translation.
Transcriptional control regulates which genes are transcribed and/or the rate at which transcription occurs.
Post transcriptional control regulates splicing, and rate of degradation of mRNA
Translational control involves how often and how rapidly an mRNA is translated.
Post translational control regulates when proteins become fully functional, how long they are functional and when they degraded.
Techniques for Producing and Analyzing DNA
- Recombinant DNA Technology: Uses Restriction endonucleases. Used in Gene cloning
- Polymerase Chain Reaction (PCR): Amplifies small samples of DNA into larger quantities so they can be tested and analysed (eg. forensics, COVID)
Recombinant DNA:
- DNA that has been prepared in the lab
- Composed of genetic material from different sources
- Purpose: To investigate genetic disorders or Production of drugs (e.g. Insulin)
Resctriction Endonucleases:
- A type of restriction enzyme
- Recognize a specific DNA sequence (target sequence) and cut the strands at a particular point within the sequence (restriction site)
- Isolated and purified only from bacteria
- Each one recognizes a different target sequence
EcoRI ex.
Sequence Specificity:
- cuts are specific and predictable
- Each enzyme cuts DNA the same way each time (produces identical fragments) – called Restriction Fragments
Staggered Cuts:
- unpaired nucleotides are left at each end of the restriction fragment
- Aka sticky ends or overhangs
- (This type of cut occurs most often)
- Can form base pairs with other single-stranded regions with a complementary sequence
blunt ends:
- Reduces specificity needed to combine sticky end fragments
- Allows any two fragments of DNA that have blunt ends to be combined
- since any two blunt ends can be combined, many by-products can form –> Less efficient
Making Recombinant DNA:
- Two DNA fragments from different sources are cut with the same restriction endonuclease to produce complementary single-stranded sticky ends
- the two cut DNA fragments are incubated with DNA ligase. seals the breaks in the DNA – forming covalent bonds between the fragments
Gene Cloning:
- Making many identical copies of a gene
- To study the gene or To use the gene to produce RNA and/or protein in larger quantities
- Bacteria mostly used in genetic engineering b/c Reproduce quickly and often, Inexpensive to maintain and Contain plasmids
Diabetes: Caused by a deficiency in production of insulin (↑ blood glucose levels). type 1 body failes to produce insulin (needs shots). type 2 body doenst produce enough or it cant use the insulin.
Dr. Frederick Banting & Charles Best isolated the gene for insulin. Helen Free invented a method to analyze blood sugar by a dip-test urinalysis. Initially, insulin was collected from the pancreas’ of pigs and cows. Caused allergic reactions in humans.
we make more insulin by Inserting human insulin gene into bacteria and bacteria makes insulin!
Gene cloning in bacteria:
Plasmids: Small, circular double-stranded DNA found in some prokaryotes, Lack a protein coat, Independent of bacterial chromosome and 1000-200,000 base pairs. Beneficial to bacteria
Carry genes that result in proteins for: Antibiotic resistance, Resistance to toxic metals, Breaking down herbicides and industrial chemicals
- Restriction endonucleases splice foreign genes into plasmids
- DNA ligase reforms phosphodiester bond between the fragments, resulting in recombinant DNA
Transformation: Introduction of foreign DNA, usually a plasmid or virus, into bacterial cells. The bacterial cells are then allowed to reproduce and transcribe/translate multiple copies of the desired gene
- Polymerase Chain Reactions:
Makes copies of desired DNA sequences (DNA photocopying)
Useful for forensic science, criminal investigations, medical diagnoses, genetic research, COVID testing
plasmids do this in gene cloning, but PCR can be used to amplify VERY SMALL quantities of DNA quickly & doesn’t rely on recombinant DNA or host systems
Usually, the DNA found at a crime scene or from a swab sample is not a sufficient quantity to test
Therefore, it must be replicated or amplified before it is cut up by restriction endonucleases and tested
This is done by the Polymerase Chain Reaction
A very small amount of DNA is required to start the process
This is necessary to study the DNA further without the risk of using up a limited sample.
DNA amplified by PCR can also be used for sequencing, or it can be cloned into a bacterial DNA for further experiments.
Like DNA replication in an organism, PCR requires a DNA polymerase enzyme
In PCR, the enzyme used is usually Taq polymerase
This is the DNA polymerase enzyme found in a heat-tolerant bacterium, Thermus aquaticus
Taq polymerase can only make DNA if it’s given a primer
PCR primers are short pieces of single stranded DNA, usually 20 nucleotides in length.
Two primers are used to flank the target region
Ingredients: Taq polymerase , primers, template DNA and nucleotides (DNA building blocks)
The ingredients are assembled in a tube and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.
Denaturation (96 degrees): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
Annealing -(55-65 degrees): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension (72 degrees) - Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
Fluctuating temps
(2^25 dna)
The cycle repeats 25-35 times and usually takes 2-3 hours.
Gel Electrophoresis:
After recombinant DNA has been isolated and amplified it can be analyzed using gel electrophoresis, which uses an electric field to separate DNA fragments by size or mass
The setup:
Agarose gel: porous matrix that DNA move through
Buffer solution: a conducting salt solution that bathes gel to carry charges
Cathode: negatively-charged electrode at the starting point of the gel to repel DNA and induce migration
Anode: positively-charged electrode at the end point of the gel to attract DNA and encourage migration
Cuts made again by endonucleases → negativ echarge dna is attracted to pos end btu also neg end → when turned on dna start to travel and gets separated → shorted small move faster and further form initial position while longer ones dont go as far and take times → overtime both types are speerated → the gel is like a filter → this is a profile of one person in 3 wells → even tho same endoculesae cuts the same thing, the location of target sequences are different so diff size fragments are produced
Identifies individuals based on their unique DNA
DNA samples from a crime scene are split into fragments using restriction endonucleases.
The fragments are separated using gel electrophoresis
Gel electrophoresis produces a unique, distinct banding pattern for each individual known as the individual’s DNA profile.
DNA profiles of different individuals can be compared to an individual of known identity to solve a crime.
We know that children inherit half their chromosomes from each parent
Therefore, they should possess a combination of parental fragments which means, all fragments produced on the child’s DNA profile must also be produced by either the mother or father.
