Chapter 4: Molecular Biology Flashcards
MCAT Mnemonics/Concepts I need to review
Why are DNA and RNA called nucelic acids?
It’s because they are found in the nucleus and possess many acidic phosphate groups
What is the dNTP?
The building block of DNA, stands for deoxyribonucleoside 5’ triphosphate where N represents one of the four basic nucleosides.
What is dATP?
Deoxyadenosine 5’ triphosphate
What makes up a nucleoside?
a sugar (deoxyribose for DNA, ribose for RNA), an aromatic, nitrogenous base, and 1-3 phosphate groups.
What are purines?
G and A
What are pyrimidines?
C, T, and U
What is the difference between a nucleoside and a nucleotide?
Nucleoside: ribose or deoxyribose w/ pyrimidine linked to the 1’ carbon.
Nucleotide: phosphate esters of nucleosides w/ 1,2, or 3 phosphate groups joined to the ribose ring by 5’ hydroxy group.
What is nucleoside triphosphate? (NTP)
When nucleotides contain 3 phosphate residues.
In individual nucleosides what is N replaced by?
A,G,C,T, or U
Mnemonic: Cut the Py
Pyrimidines: C, U,T
What is the backbone of DNA?
The sugar + phosphate portion of the nucleotide (invariant or unchanging) while the nitrogenous base is the variable portion of the building block.
What are the aromatic nitrogenous bases? Classify each as a purine or pyrimidine.
Adenine - purine
Cytosine - pyrimidine
Thymine - pyrimidine
Uracil - pyrimidine
Guanine - purine
What links nucleotides together to form polynucleotides and make up the DNA?
How specifically are two nucleotides bonded?
Phosphodiester bonds
3’ OH group of one deoxyribose bonds to the 5’ phosphate group of the next deoxyribose
What is the difference between an oligonucleotide and a polynucleotide?
Oligonucleotide = only several nucleotides linked together
Polynucleotide = many nucleotides linked together
What is the Watson-Crick Model?
Cellular DNA is right right-handed double helix held together by hydrogen bonds between bases. The strands run antiparallel
What is Chargoff’s Rule?
A=T
G=C
A+G = C+T
How many Hydrogen bonds between bases in a dsDNA?
AT = 2 H bonds
GC = 3 H bonds
A hydrogen-bonded pair always consists of a ____ plus a ______
purine plus a pyrimidine
What does annealing/ hybridization mean?
The binding of two complementary strands of DNA into a double stranded structure
What does melting/ denaturation mean?
The separation of two complementary strands of DNA from the double stranded structure
Which of the following is/are true about dsDNA?
I. If the amount of G in a double helix is known, the amount of C can be calculated
II. If the fraction of purine nucleotides and the total molecular weight of a double helix are known, the amount of cytosine can be calculated
III. The two chains in a piece of dsDNA containing mostly purines will be bonded together more tightly than the two chains in a piece of dsDNA containing mostly pyrimidines
IV. The oligonucleotide ATGTAT is complementary to the oligonucleotide ATACAT
I, IV are true
I is true because for every G there is a C and for every A there is a T.
II is false because the ratio of purines to pyrimidines is always 50:50 since a purine is always paired with a pyrimidine. To calculate the amount of any base you have to know the AT and GC pairs..
Item III is false because the ratio of purines to pyrimidines is always the same. However, two chains containing mostly GC pairs will bind more tightly than two chains containing mostly AT pairs, since GC are held together by 3 H bonds while AT pairs only have 2.
IV is true because the 5’ end is always written first unless specified otherwise, and the strands are antiparallel: A and T pair, G and C pair.
What is a genome?
The sum total of an organism’s genetic information
What is a chromosome and how many do humans have?
A chromosome is a large piece of linear dsDNA that make up the eukaryotic genome
Humans have 46 chromosomes (23 pairs, one from each parent)
What is the difference between eukaryotic, prokaryotic, and viral genomes?
eukaryotic contains chromosomes
prokaryotic contains one circular chromosome
viral can be linear or circular DNA or RNA
How many base pairs does the human genome have vs the bacterial genome?
humans have 10^9
bacterial have 10^6
What is DNA gyrase? Describe the steps.
An enzyme that requires ATP to twist the circular DNA in prokaryotes
Breaks the DNA and twists the two sides of the circle around each other, resulting in a twisted circle that is composed of dsDNA
Creation of supercoils
What are histones?
Globular proteins where DNA is wrapped around it
What are nucleosomes?
Made up of an octamer of histones with DNA wrapped around it
What is linker DNA?
The string-like DNA between nucleosomes that is bound by a single linker histone
What is a chromatin?
Heterochromatin?
Euchromatin?
Fully packed DNA that is composed of closely stacked nucleosomes
When staining, it is the darker region that is more dense and rich in repeats
When staining, it is the lighter region that is less dense and therefore has higher transcription rates and higher gene activity because the looser packing makes DNA accessible to enzymes and proteins
What is the centromere?
What is it made of?
The region of the chromosome that spindle fibers attach to during cell division
Made of heterochromatin and repetitive DNA sequences
What is a kinetochore?
Multiprotein complexes that act as anchor attachment sites for spindle fibers
What types of arms do chromosomes have?
What are the different centromere positions called? Name and describe each.
short arm (p)
long arm (q)
- Metacentric - p and q are equal
- Submetacentric - q is slightly longer than p
- Acrocentric - q is a stalk while p is bulb-like
- Telocentric - there is only one arm because the centromere is at the end of the chromosome
What is a telomere? Be specific.
The ends of linear chromosomes that have repeated nucleotide sequences (about 50-several hundred times)
Can be single or double stranded DNA about 300 BP long
Acts as a disposable buff that blocks the ends of chromosomes to prevent deterioration and also prevents fusion with neighboring chromosomes
On average, how long is one repeated unit in telomeres and what is it rich in?
6-8 BP long repeated unit
Guanine rich
How many BP is the human genome?
3.2 billion BP
How many genes does the human genome code for?
21,000
What are intergenic regions?
What is their purpose?
What are some important things in the intergenic region?
Non-coding DNA
Can direct assembly of specific chromatin and can contribute to regulation of nearby genes
Intergenic regions includes tandem repeats and transposons
What is SNP’s? Give details.
How often does it occur?
Single Nucleotide Polymorphisms which are single nucleotide changes/ mutations that mostly occur in the non-coding regions of the genome. However, some SNPs lead to specific traits and phenotypes.
Occurs once in every 1,000 BP
If the size of the human genome is just over 3 billion base pairs, approximately how many human SNPs are there?
3 mill base pairs are human SNPS
Since SNPs happen once every 1000 BPs
3 billion BP / 1000 BP = 3 mill human SNPs
What is CNV?
Misalignment of repetitive DNA sequences during synapsis of homologous chromosomes in meiosis leads to different copies of DNA.
How big is a region of CNV?
10^3 to 10^6 BP
Where is CNV commonly seen?
0.4% of the genome
Associated with cancer and other diseases
Immune system function
Brain development and activity
What is a tandem repeat?
short sequences of nucleotides repeated (3-100 times) one after the other
What constitutes as an unstable tandem repeat?
What are the effects of an unstable tandem repeat?
When the repeating unit is short (di- or tri-nucleotides) or when the repeat itself is very long
Unstable tandem repeats can lead to chromosome break and disease
What is transcription and what can it make?
Reading DNA and writing the information as RNA
Can make a final gene product (all non-coding RNA)
Can make a messenger molecule
What is translation? What enzyme does it involve?
The synthesis of proteins using mRNA as a template
Ribosome is the massive enzyme that is composed of many proteins and rRNA
____ encodes and transmits the genetic info passed down from parent to offspring not _____
1) DNA
2) protein
What is the Central Dogma?
Inherited information used to create enzymes and structural proteins
DNA –> RNA –> proteins
What is the genetic code?
The language used by DNA and mRNA
A,G,T,C
What is a codon?
A 3 letter nucleotide sequence that codes for a particular amino acid.
How many possible different codons are there and how can that be calculated?
64 codons (enough to specify 20 unique amino acids)
4 nucleotides
3 nucleotides per codon
4^3 = 64 different combinations
Why is uracil shown in the chart and why is thymine absent? (pg 74)
This is because RNA is the nucleic acid that encodes protein during translation. RNA has a U instead of a T.
The codon GTG in DNA is transcribed in RNA as _____, which the ribisome translates into what amino acid?
G =C
T= A
G= C
The RNA codon was transcribed from DNA as CAC coding for histidine.
What is a stop codon?
What is another name for it?
Does not code for a specific amino acid, but instead notifies the ribosome that the protein is complete. Causes it to stop reading the mRNA
Also called nonsense codons
If the last nucleotide in the codon CUU is changed in a gene that codes for a protein, will the protein be affected?
No since CUN codes for leucine, regardless of what N is. Notice that switching the third nucleotide in the majority of codons will have no effect.
What are synonyms for the genetic code and how does this affect the genetic code?
Synonyms are when two or more codons code for the same amino acid
The genetic code is degenerate because of this
What are some examples of molecular biology that is outside of the Central Dogma?
Retroviruses make DNA from RNA using the enzyme reverse transcriptase.
Info can be transferred in other ways such as DNA methylation and post-translational modification of proteins that can alter gene expression and convey information, even though they are not directly included in the Central Dogma.
Lastly, many final products are not proteins but rather RNA instead.
What is the Meselson-Stahl Experiment?
Semiconservative replication - One strand of the DNA is parental while the other strand is the daughter strand
Conservative replication - the parental dsDNA stays as is while an entirely new ds genome is created as the daughter
Dispersive replication - both copies of the genomes were composed of scattered pieces of new and old DNA
DNA replication is _____
semiconservative
Individual strands of the double-stranded parent are pulled apart and then a new daughter strand is synthesized using the parental DNA as a template to copy from. Each new daughter chain is perfectly complementary to its template or parent.
What is helicase?
An enzyme that requires ATP to break the hydrogen bonds and separate the dsDNA at the origin of replication
How do prokaryotes find the ORI?
Eukaryotes?
Prok: DnaA protein finds the ORI
Euk: three proteins cooperate to find the ORI
2 of the proteins are synthesized during M and G1 phase and destroyed in the S phase. These link DNA replication to the cell cycle to ensure DNA replication doesn’t initiate during other phases of the cell cycle
What is topoisomerase?
An enzyme that cuts one or both of the strands and unwraps the hel
What is SSBP?
Single stranded binding protein that protects the DNA that has been unpackaged in preparation for replication and to help keep the strands separated
What is an open complex?
Where the strands are separated during DNA replication
What is a primase?
An RNA polymerase that is part of the primosome. It synthesizes an RNA primer for each template strand during DNA replication
What is a primosome?
A set of proteins that help to synthesize an RNA primer for each template strand during DNA replication
How long is the average RNA primer?
8-12 nucleotides long
What is DNA polymerase?
Elongates the primer on the daughter strand by adding dNTP’s to its 3’ end
3’ OH group acts as a nucleophile in the polymerization reaction to displace 5’ pyrophosphate from the dNTP to be added.
What direction is the template strand read in?
3’ to 5’ because the daughter strand is made in the 5’ to 3’ direction and the two strands have to be antiparallel
What is a replisome?
A large complex of proteins that help DNA polymerase polymerize the DNA quickly
How many components does a prokaryotic replisome have?
Eukaryotic? Why?
13 components
27 components because it must also unwind the DNA from the histones
What is the driving force for the polymerization reaction?
Removal and hydrolysis of pyrophosphate (P2O7 4-) from each dNTP added to the chain
What direction does polymerization always happen in?
5’ to 3’ direction without exception
This means the existing chain is always lengthened by the addition of a nucleotide to the 3’ end of the chain. There is never 3’ to 5’ polymerase activity.
What does DNA polymerase require?
A template and a primer
It cannot make a DNA chain from scratch but must copy an old chain. This makes sense b/c it would be useless if DNA pol just made a strand of DNA randomly without copying a template.
Needs a primer since it cannot start a new nucleotide chain
Can DNA polymerase make the following partially double-stranded structure completely double-stranded in the presence of excess nucleotides using the top strand as a primer? (pg. 78)
No. The DNA strands are antiparallel meaning that the upper strand would have to be extended in a 3’ to 5’ direction which is impossible. (excess nucleotide is an extraneous phrase, typical MCAT smokescreen)
What is a replication fork?
The area where the parental double helix continues to unwind during DNA replication
Replication proceeds in both directions away from the origin of replication. Both template strands are read ___ to ___ while daughter strands are elongated ____ to ____
1) 3’ to 5’
2) 5’ to 3’
What is a leading strand?
Lagging strand?
Elongating continuously
Needs RNA primers constantly as the replication fork widens
What are Okazaki fragments?
Small chunks of DNA on the lagging strand
As the replication forks grow, does helicase have to continue to unwind the double helix and separate the strands?
Yes
Replication forks grow ____ from the origin in both directions. Each replication fork contains an _____ and a _____
1) away
2) leading strand
3) lagging strand
Replication of the leading strand is ____
continuous and leads into the replication fork
Replication of the lagging strand is ____
discontinuous
resulting in Okazaki fragments
Eventually all RNA primers are replaced by ____
DNA
What are the Okazaki fragments joined by?
DNA ligase, an enzyme
Why is DNA polymerase considered processive?
Because it can rapidly build DNA and is able to add tens of thousands of nucleotides before falling off the template
Where is DNA Polymerase I-V found?
Describe the purpose of each DNAP
(DNA III and I are most important)
Prokaryotes
I: Slow processivity allows it to start adding nucleotides onto the RNA primer, but DNAPIII takes over about 400 BP downstream. This means it has 5’ to 3’ polymerase activity. Also has 3’ to 5’ exonuclease proofreading activity. In addition, 5’ to 3’ exonuclease activity that removes the RNA primer and replaces it with DNA in a 5’ to 3’ polymerase activity. Lastly, it is important for excision repair
II: Used as a back up for DNAPIII so it has 5’ to 3’ polymerase activity as well as 3’ to 5’ exonuclease proofreading activity, and is involved in DNA repairing pathways
III: High processivity means that there is fast and accurate 5’ to 3’ polymerase activity. Known as a replicative enzyme because it has no known function in repair. Has 3’ to 5’ exonuclease proofreading activity
IV and V: Error prone in 5’ to 3’ proofreading activity, but functions to stall other polymerase enzymes at replication forks when DNA repair pathways have been activated
What happens during 3’ to 5’ exonuclease activity?
What is another name for this?
The enzyme moves backwards to chop off the nucleotide it just added, if it was incorrect
Proofreading function
What is the difference between an exonuclease and an endonuclease?
Exonuc: cutting a nucleic acid chain at the end
Endonuc: cutting a polynucleotide acid chain in the middle of the chain, usually a particular sequence
If a bacterium possesses a mutation in the gene for DNA polymerase III, resulting in an enzyme without the 3’ to 5’ exonuclease activity, will mutations occur more often than in bacteria with normal DNA polymerase gene?
Yes, The 3’ to 5’ exonuclease activity is the polymerase’s way of editing its work. Without this editing function, many more point mutations would occur due to the incorporation of wrong nucleotides. The normal polymerase is remarkably adept at sensing correct base paring and removing bases that don’t belong.
What are some examples of an endonuclease? List and describe.
Repair enzymes remove chemically damaged DNA from the chain
Restriction enzymes are found in bacteria and they destroy the DNA of infecting viruses, thereby restricting the host range of the virus
What is theta replication/ theta mechanism?
Replication that occurs in prokaryotes because it only has one circular chromosome and one origin, so a partially duplicated genome looks like the Greek letter theta
How many origins may a eukaryotic replication have, and why?
Many have many origins because having only one would be too slow aka replication bubbles
What are telomeres?
Disposable repeats at the end of chromosomes that are consumed and shorted during cell division (becomes between 50-200 BP shorter)
When telomeres become too short, they reach a critical length. What are some possible paths it can take following this occurrence?
Enters a senescent state where the cell is alive but no longer dividing
Activates DNA repair pathways
Activates apoptosis, or pre-programmed cell death
What is the Hayflick limit?
The number of times a normal human cell type can divide until telomere length stops cell division
What is a telomerase?
An enzyme that adds repetitive nucleotide sequences to the ends of chromosomes and lengthens telomeres.
A ribonucleoprotein complex that contains RNA primer and reverse transcriptase that reads RNA templates and generates DNA
Only expressed in the germ line, embryonic stem cells, or some white blood cells in most organisms
Cancer cells can also express telomerase, which helps the cancer cells to immortalize
What is a germ-line mutation?
A mutation that can be passed onto offspring since it occurs in the germ cells which give rise to gametes
What is a somatic mutation?
A mutation that is not passed onto offspring since it occurs in somatic/ non-gametic cells
Generally, what are the causes of mutation? (broad concept)
Environmental or chemical factor as well as occurring spontaneously
What are examples of ionizing radiations and what affect does it have on DNA?
X-ray, alpha particles, gamma rays
Causes DNA breaks
What affect does UV light have on DNA?
Give a specific example.
Causes a photochemical damage to DNA
Pyrimidine dimer formation when two pyrimidines (2 C’s or 2 T’s) are close to each other and covalently linked to each other, which causes a distortion in the DNA backbone that can cause mutations during DNA replication if it isn’t repaired
What is a mutagen?
Any compound that can cause mutations
What is an intercalator?
What is an example of an intercalator?
Compounds that look like purines and pyrimidines with large flat aromatic ring structures that cause mutations by inserting themselves between base pairs
Ethidium Bromide
What are lysogenic viruses?
Viruses that insert into the genome of the host cell and disrupt genetic function
What are the 7 kinds of mutations?
1) Point mutations
2) Insertions
3) Deletions
4) Inversions
5) Amplifications
6) Translocations and rearrangements
7) Loss of heterozygosity
What are the three types of point mutations? List and describe each.
1) Missense - One amino acid is replaced with a different amino acid. If the amino acids are similar it may not be serious
2) Nonsense - A stop codon replaces a regular codon and prematurely shortens the protein
3) Silent - A codon is changed into a new codon for the same amino acid, so there is not change in the protein’s amino acid sequence
In a missense mutation, how can the replacement of one amino acid for a similar amino acid occur and not be serious?
For example, substitution a small hydrophobe (valine) for another small hydrophobe (leucine) will cause little disruption of the protein structure.
They cause changes in primary structure but do not affect secondary, tertiary, or quaternary structure
What is a frameshift mutation and in what ways can you have a frameshift mutation? List and describe
Mutations that cause a change in the reading frame
Insertions and deletions can result in a frameshift mutation where there is the addition or deletion of nucleotides to or from the DNA sequence
Are all insertions and deletions frameshift mutations?
No because if a whole codon is inserted or deleted then an amino acid is added or deleted without changing the reading frame
What are inversions?
A type of mutation where a segment of a chromosome is reversed end to end. The chromosome undergoes breakage and rearrangement within itself.
What are transitions in point mutations?
substitution of a pyrimidine for another pyrimidine
What are transversions in point mutations?
Substitution of a purine for a pyrimidine
What is an amplification?
A type of mutation where the segments of a chromosome are duplicated
What is translocation and rearrangement?
A type of mutation that occurs when recombination happens between non-homologous chromosomes that can create a gene fusion, where a new gene product is made from parts of two genes that were not previously connected
What is balanced translocation?
Unbalanced?
Where no genetic information is lost
Where genetic information is lost or gained
What is a transposon?
What types of mutations can it cause?
A mobile genetic element
Can cause inversions, deletions, and rearrangements
What makes up most eukaryotic transposons?
Degenerate retroviruses
What are three types of transposons? List and describe
1) IS elements are composed of a transposase gene flanked by inverted repeat sequences
2) Complex transposons are transposons that contain genes like antibiotic resistance
3) Composite transposons are two similar or identical IS elements with central region in between
What is a transposase?
A protein that is coded for by transposons, and has cut-and-paste activity where it catalyzes mobilization of the transposon. Acts as the scissors
Sometimes the transposon sequence is _____ _____ and moved, and sometimes it’s _____ and moved; ____ _____ are important for this mobilization
completely excised
duplicated
Inverted repeats
Many mobilizations have no effect because ….? BUT some can cause mutations if ….?
…. it inserts into a relatively unimportant part of the genome
…. they jump into an important part of the genome
If the transposon jumped into a:
…promotor
….protein coding region
….regulatory part of the genome
then what happens to the genome respectively for each event?
… turns of gene expression
….. disrupts/ mutates the sequence
….. ramps up gene expression at a nearby site
Transposons can cause structural changes to chromosomes when what happens?
When they work in pairs
If a chromosome has two transposons with the same direction, then what happens?
…And if the two transposons were pointing in opposite directions?
Chromosomal segment between the two transposons loop around in order for the transposons to line up parallel to each other. Recombination happens and deletion of the DNA between the transposons happens. The DNA that is lost takes one transposon with it, meaning it can jump back into the genome somewhere else, causing chromosome rearrangement
Chromosomal segment between the two transposons form a hairpin like structure so that the transposons can line up parallel to each other. Recombination happens and the DNA between the two transposons ends up inverted
What is loss of heterozygosity?
A type of mutation where a deletion removes the normal copy of the gene and the only remaining copy is the mutated version. Makes the locus hemizygous, meaning there is only one gene copy in the diploid organism
What does it mean to be hemizygous?
Only having one gene copy in the diploid organism
Do mutations on sex chromosomes have a greater or lesser effect than the ones on autosomes? Why?
Greater effect because autosomes are present in double copies so a mutation can be easily repaired if the other copy is normal
What is a gain-of-function mutation?
Increases the activity of a certain gene product, or changes it so that it gains a new and abnormal function
What is a loss-of-function mutation?
Results in the gene product having less or no function
What is Haploinsufficiency?
When a diploid organism has a single functional copy of a gene and it’s not enough to support a normal state
What are two examples of beneficial mutations in humans? List and describe.
Sickle-cell Anemia is when deoxygenated hemoglobin dimerizes and form long chains that cause the RBC to sickle, then are prematurely destroyed, and leads to anemia. People who carry this gene are more resistant to malaria and that’s why it’s beneficial
35 BP are missing from the CCR5 gene. HIV resistance is given to homozygotes and delays AIDS onset in heterozygotes
What are Inborn errors of metabolism?
What is the cause and what are the symptoms?
A huge group of genetic diseases that involve disorders of metabolism.
Cause: Single mutation in a single gene that codes for some sort of metabolic enzyme
Symptom: Build-up of a toxic compound that can’t be broken down OR by defeciency of an essential molecule that can’t be synthesized, as well as many other symptoms regarding their respective metabolic pathway that the mutation affects
What is cancer driven by?
Mutation accumulation
What are the two things that can cause cancer?
Can be inherited or caused by carcinogen exposure
What is a carcinogen?
A mutagen that is directly involved in causing cancer
Tumors have mutations that are in ____ and ____ _____
oncogenes
Tumor suppressors
What are oncogenes?
Genes that can cause cancer when it’s mutated or expressed at high levels
What are tumor suppressor genes?
If they are deleted or expressed at low levels, it can cause cancer
What is photoreactivation?
What organism(s) is it used in?
Involved in direct reversal in DNA repair when an enzyme uses visible light to repair UV-induced pyrimidine photodimers.
Commonly performed by bacteria and many plants
What is Homology-dependent Repair?
What types of repair are homology-dependent? List and describe.
A type of repair that is dependent upon the DNA characteristic that DNA is double-stranded and complementary
1) Excision repair is repair that happens before DNA replication. It removes defective bases or nucleotides and replaces them. If it doesn’t, then replication machinery can’t pair them properly
2) Post-replication repair is repair that happens during and after DNA replication
What is a type of post-replication repair? Name and describe
Mismatch repair pathway (MMR) that targets mismatched Watson-Crick BP that weren’t repaired by DNA polymerase proofreading during replication. To figure out which base is the correct one in bacteria, it uses methylation on the parental template strand. In other prokaryotes and most eukaryotes, the newly synthesized strand is recognized by the free 3’ terminus on the leading strand, or by the presence of gaps between Okazaki fragments on the lagging strand
What may cause a double strand break in DNA?
1) Reactive oxygen species
2) Ionizing radiation
3) UV light
4) Chemical agents
What are two types of DSB repair? List and describe their processes
1) Homologous recombination happens when the DSB is identified and nuclease trims at 5’ ends and breaks the phosphodiester bonds, then the helicase unwinds the DNA to generate ssDNA, and many proteins bind to the ends to search for a sister chromatid region that’s complementary
2) Non-homologous end-joining is for cells that aren’t actively growing or cycling through the cell cycle because it doesn’t have the option of using sister chromatids to repair the DSB in and error-free way. It does not require specificity; the broken ends are stabilized and processed, and then DNA ligase connects the fragments. This can result in BP’s being lost or chromosomes being connected in an abnormal way.
What is gene expression?
The process where information contained in genes begins to have effects in the cell
What are the three differences between RNA and DNA?
RNA is :
single stranded, except in some viruses
contains uracil instead of thymine
the pentose ring in RNA is ribose rather than 2’ deoxyribose
What are the 3 parts of a coding RNA?
The 5’ UTR or untranslated region that is not translated into protein
The ORF or open reading frame that codes for a protein and starts at the start codon and ends at a stop codon
The 3’ region after the stop codon that isn’t translated into protein but contains regulatory regions that influence post-transcriptional gene expression
What is monocistronic and in what organism is it found?
mRNA that has only one ORF that codes for one polypeptide, and thus one protein. Follows the “one gene, one protein” principle.
Found in Eukaryotes
What is polycistronic and in what organism is it found?
mRNA that codes for more than one polypeptide, and has more than one ORF. The different ORFs are generally related in function
fond in Prokaryotes
Where are the initiation, translation, and termination sequences found in a polycistronic mRNA?
Describe what the termination and initiation information do.
Between ORFs
Termination information helps finish the PREVIOUS peptide chain
Initiation helps start translation of the NEXT ORF on the transcript
What is hnRNA? Give details
What organism is it found in, and why?
Heterogeneous nuclear RNA is the RNA transcribed from DNA that is immature and is a precursor to mRNA.
ONLY found in eukaryotes because prokaryotes don’t go through processing events
What is mature mRNA?
when hnRNA goes through splicing, and addition of the methyl cap and the poly A tail, it then becomes mature and ready to be translated
What is ncRNA?
Non-coding RNA that isn’t translated into a protein but is still functional
What are some of the most common types of ncRNA? List and describe.
1) Transfer RNA (tRNA) carries the amino acid from the cytoplasm to the ribosome to be added to a growing protein
2) Ribosomal RNA (rRNA)
is the major component of the ribosome.
Humans only have four types of rRNA (18S,5.8S,28S, and 5S) but almost all RNA made in a given cell is rRNA. Serves as components of the ribosome.
Estimate how many different tRNAs there are.
between 20-61 tRNAs
What are ribozymes?
catalytic RNAs, they are capable of performing specific biochemical reactions similar to protein enzymes.
What is small nuclear RNA (snRNA)?
molecules (150 nucleotides) associate with proteins to form sRNP (small nuclear ribonucleic particles) complexes in the splicesome.
MicroRNA (miRNA) and small interfering RNA (siRNA)
function in RNA interference (RNAi) a form of post-transcriptional regulation of gene expression. Both can bind to specific mRNA molecules to increase or decrease translation.
What are PIWI-interacting RNAs (piRNAs)?
Are single-stranded and short (typically between 21 and 31 nucleotides in length). They work with a class of regulatory proteins called PIWI proteins to prevent transposons from mobilizing.
What are long ncRNAs?
are longer than 200 nucleotides. They help control basal transcription levels in a cell by regulating initiation complex assembly on promoters. They also help contribute to many types of post-transcriptional regulation by controlling splicing and translation and they function in imprinting and X-chromosome inactivation.
What is transcription?
the synthesis of RNA (mRNA, tRNA, or rRNA) using DNA as the template. Information is transferred from one polynucleotide to another.
RNA transcript produced in transcription is ___ to DNA template just as the daughter strand produced in replication was.
complementary
Replication vs Transcription: Similarities
The driving force for both processes is hte removal and subsequent hydrolysis of pyrophosphate from each nucleotide added to the chain with the existing chain acting as a nucleophile.
Transcription can only occur in the 5’ to 3’ direction
Begins at a specific spot on the chromosome.
Replication vs Transcription: Differences
RNA pol does not require a primer.
RNA pol can’t remove mismatched nucleotides (no exonuclease activity) so it can’t correct its errors.
Lower fidelity process than replication.
DNA replication begins at the origin
RNA transcription begins at the start site.
Only ___ of the strands of the DNA template encodes a particular mRNA molecule.
one
Paired DNA strands are ___ not ___
complimentary not identical
What is the strand that is actually transcribed called in RNA transcription?
template, non-coding, transcribed, antisense strand. Its complimentary to the transcript
What is the other strand in RNA transcription called?
Coding or sense strand (has same sequence as transcript except the T is a U).
Downstream vs upstream?
Downstream: toward the 3’ end of the coding strand and transcript (+ #s)
Upstream: towards the 5’ end of the coding strand, beyond the 5’ end of the transcript
(-#s)
Describe the elements of prokaryotic transcription including the core enzyme.
All types of RNA are made by the same RNA polymerase.
5 subunits: 2 alpha, 1 beta, 1 beta’, 1 omega.
Omega is the core enzyme responsible for rapid elongation of the transcript but needs sigma factor to make a holoenzyme that helps with initiation.
What are the three stages of prokarytoic transcription?
Initiation: RNA pol holoenzyme binds to the promoter (two primary sequences Pribnow box at -10 and -35 sequences). Holoenzyme scans along the chromosome like a train on a railroad track until it recognizes a promoter and then stops, forming a closed complex. The RNA pol must unwind a portion of the DNA double helix before it can begin to synthesize RNA. The RNA pol bound at the promoter with a region of a single-stranded DNA is termed the open complex. Once the open complex forms, transcription can begin.
Elongation: The core enzyme elongates the RNA chain processively with one polymerase and complex synthesizing an entire RNA molecule. As the core enzyme elongates the RNA, it moves along the DNA downstream in a transcription bubble in which a region of the DNA double helix is unwound to allow the polymerase to access the complementary DNA template.
Termination: When the termination signal is detected (with the help of rho) the polymerase falls off the DNA, releases the RNA and the transcription bubble closes.
What are the roles of the sigma factor?
Help the polymerase find promoters by:
1) Increasing the ability of RNA polymerase to recognize promoters.
2) Decrease the nonspecific affinity of holoenzyme for DNA.
Once open complex and several phosphodiester bonds are formed, the sigma factor isn’t needed and it leaves the RNA pol complex
Prokaryotic vs. Eukaryotic Transcription
Prokaryotic:
-before the kernel/karyon (nucleus) b/c they have no nucleus so transcription occurs free in the cytoplasm, the same area where translation occurs meaning both can happen simultaneously.
-The primary transcript in prokaryotes is mRNA.
Eukaryotic:
-Must transcribe their mRNA in the nucleus, modify it, and then transport it across the nuclear membrane to the cytoplasm where it can be translated. Transcription and translation DO NOT occur simultaneously.
- Eukaryotic primary transcript hRNA made by RNA pol II is modified a ton before translation.
What are introns?
These are intervening sequences in the RNA.
Avg size is about 2000 nucelotides
What are exons?
Protein coding regions of the RNA because they get expressed.
Before the RNA can be translated introns must be ___ and exons ____ this is done by _____
1) removed
2) joined together
3) splicing
What is a splicesome?
A complex containing over 100 proteins and 5 small nuclear RNA (sRNA) molecules and snRNPs. It assembles around the introns that need to be removed.
How does splicing occur?
To catalyze the splicing reaction, snRNPs recognize and hydrogen bond to conserved nucelotides in the intron: usually GU at the 5’ end, AG at the 3’ end, and adenine 15-45 bases upstream of 3’ splice site. This aligns the hnRNA so that splicing can take place.
Two splicing reactions are catalyzed by splicesome.
First rxn attaches one end of intron to conserved adenine causing it to make a loop, then the second rnx joins two exons and releases the loop. The five conserved nucleotides needed for this rxn are found in all eukaryotic species.
What are the 5 conserved nucleotides needed for splicing?
GU, A, and AG are found in all genes and across all eukaryotic species.
What is alternative splicing?
Alternative splicing is a cellular process in which exons from the same gene are joined in different combinations, leading to different, but related, mRNA transcripts. These mRNAs can be translated to produce different proteins with distinct structures and functions — all from a single gene.
What is a 5’ cap?
methylated guanine nucleotide stuck on the 5’ end. Also prevents the digestion of mRNA by exonucleases that are free in the cell.
What is a poly-A tail?
string of several hundred adenine nucleotides. Essential for translation and also prevents the digestion of mRNA by exonucleases that are free in the cell.
What are the different eukaryotic RNA pol?
RNA poly I transcribes most rRNA
RNA poly II transcribes hnRNA (mRNA), most snRNA, and some miRNA
RNA pol III trascribes tRNA, long ncRNA, siRNA, and some miRNA and a subset of rRNA
What is translation?
This is the synthesis of polypeptides according to the amino acid sequence dictated by the sequence of codons in the mRNA.
1) An mRNA molecule attaches to a ribosome at a specific codon, and the appropriate amino acid is delivered by a tRNA molecule. Then the second amino acid is delivered by another tRNA molecule. Then the ribosome binds the two amino acids together creating a dipeptide. This is repeated until the polypeptide is complete at which point the ribosome drops the mRNA and the new polypeptide departs.
What is an anticodon?
It is a sequence of 3 ribonucleotides which are complementary to the mRNA codon the tRNA translates.
What specificity dictates which amino acid of the twenty will be added to a growing polypeptide chain by the ribisome?
The specific base paring between the tRNA anticodon and the mRNA codon.
What is the amino acid acceptor site?
This is where the amino acid is attached to the tRNA.
Key point: There is a tRNA for each codon so each tRNA is specific for one amino acid.
Meanwhile, each amino acid may have several tRNAs.
What is the Wobble Hypothesis?
Most organisms have fewer than 45 different types of tRNAs meaning some anticodons must pair with more than one codon.
The hypothesis explains that the first two codon-anticodon pairs obey normal base pairing rules but the third position is more flexible, allowing for non-tradiational pairing, and explains why a smaller number of tRNAs are possible.
What is a particularly wobbly combo?
A modified inosine base (I) at the 5’ end of the anticodon, it can bond to 3 different codon bases including A,U, or C.
What are the most common wobble base pairs?
G-U, I-U, I-A, I-C.
5’ Base in Anticodon (tRNA)
G
C
A
U
I
3’ Base in Codon (mRNA)
C (WCB) or U (WB)
G
U
A (WCB) G (WB)
A, U, or C (all WB)
What is tRNA loading/amino acid activation, what are the steps, and why is it useful?
2 high-energy phosphate bonds are hydrolyzed to provide the energy to attach an amino acid to its tRNA molecule.
Steps:
1) AA attached to AMP to form aminoacyl AMP. Nucleophile = acidic oxygen of AA LG is PPi
2) The pyrophosphate leaving group is hydrolyzed to 2 orthophosphates. Highly favorable rxn G«_space;0
3) tRNA loading an unfavorable reaction is driven forward by destruction of the high energy aminoacyl-AMP bond created in Step 1.
Eventually, the bond between the AA and tRNA molecule will be broken. This hydrolysis will power the peptide bond formation: the nitrogen of another amino acid will nucelophillically attack the carbonyl carbon of this AA and the tRNA will be the LG
Requires: 2 ATP equivalents b/c it uses 2 high-energy bonds.
Useful b/c breaking aminoacyl tRNA bond will drive peptide bond formation forward.
What is aminoacyl-tRNA synthase enzymes?
These enzymes are specific to each amino acid. They are highly specific and recognize each amino acid-base and tRNA by 3D structure.
What is the function of amino acid activiation?
Specific and accurate amino acid delivery and the other is thermodynamic activation of amino acid.
Describe the ribisome
Composed of many polypeptides and rRNA chains held together in a big quaternary structure. They float around in the cytoplasm and have a small subunit and a large subunit.
What is the A site?
aminoacyl-tRNA is where each new tRNA delivers its amino acid
What is the P site?
peptidyl-tRNA site is where the growing polypeptide chain still attached to a tRNA is located during translation.
What is the E site?
exit tRNA is where the now empty tRNA sits prior to its release from the ribisome.
How do the tRNAs move through the 3 sites?
A>P>E
tRNAs = APE
How does prokaryotic translation work?
Translation occurs in the same compartment at the same time as transcription. While the mRNA is being made ribosomes attach and begin translating it.
What are the differences between the prokaryotic and eukaryotic translation (summary)
- The ribosome is larger (80S) with/ different components.
- mRNA must be processed before it can be translated (spliced w/ cap and tail added)
- N-terminal amino acid is different (Met vs fMet).
Eukaryotic mRNA requires transport from nucleus to cytoplasm, thus transcription and translation cannot proceed simultaneously.
What is cap-dependent translation?
In eukaryotic translation, the important role of 5’ end mRNA cap recognition as most eukaryotic transcripts were monocistronic and coded for only one polypeptide chain
What is cap-independent translation?
when eukaryotic translation occurs in the middle of an mRNA molecule.
Must have an internal ribosome entry site or IRES. Most code for proteins that help the cell deal with stress.
What is epigenetics?
Changes in gene expression that are not due to mutations, but are long-term and heritable. (DNA methylation, chromatin remodeling, and RNA interference.
What is DNA methylation and how does DNA methylation work in prokaryotes vs eukaryotes?
DNA methylation is covalent modification through the addition of a methyl group. Bacteria methylate new DNA shortly after synthesis and brief delay is good in mismatch repair pathways.
Prokaryotes: control gene expression by promoting or inhibiting transcription.
Eukaryotic:
controls gene expression (especially in development) and is implicated in several diseases.
1) Methylation physically blocks genes from transcriptional proteins
2) Certain proteins bind methylated CpG groups ad recruit chromatin remodeling proteins that change the winding of DNA around histones.
Repressible vs inducible enzymes
Anabolic enzymes whose transcription is inhibited in the presence of an excess amount of product are repressible.
Catabolic enzymes whose transcription can be stimulated by the abundance of substrates are called inducible enzymes.
Lac operon vs trp operon
Lac operon: inducible since the enzymes it codes for are part of lactose catabolism.
trp operon: is repressible since the enzymes it codes for mediate tryptophan biosynthesis or anabolism.
What makes up an operon?
1) coding sequence for enzymes
2) upstream regulatory sequences or control sites.
May also include genes for regulatory proteins like repressors or activators but not required.
What makes up the lac operon?
1)P region: promoter site on DNA to which RNA pol binds to initiate transcription of Y, Z, and A genes.
2) O region: the operator site to which the Lac repressor binds
3) Z gene: codes for enzyme Beta-galactosidase which cleaves lactose into glucose and galactose.
4) Y gene: codes for permease, a protein that transports lactose into the cell.
5) A gene: codes for transacetylase an enzyme which transfers an acetyl group from acetyl-CoA to Beta galactoside.
What two genes code for proteins important in regulating the lac operon?
1) crp gene: located at the distant site, codes for catabolite activator protein (CAP) and helps couple lac operon to glucose levels in the cell.
2) I gene: located at a distant site, gene codes for Lac repressor protein.
What happens when RNA Pol cannot bind the Promoter in the presence of Lac Repressor protein? (Lac Operon in the presence of glucose and absence of lactose)
Result: Z, Y, and A are not transcribed or translated.
What happens when the RNA pol binds the promoter weakly? (The lac operon in the presence of both glucose and lactose)
Result: Z,Y, and A are transcribed at low levels.
In high glucose conditions, adenyl cyclase is ___, and cAMP levels are ___ (and vice versa)
1) inactivated
2) low
What happens when the RNA pol binds the promoter strongly (lac operon in absence of glucose and presence of lactose)
Result: Z, Y, and A are transcribed at high levels
What happens when RNA pol cannot bind the promoter in the presence of tryptophan? (The Trp operon in the presence of tryptophan)
ResultL: trp genes are off in the presence of tryptophan
