Molec and Cell 5 Flashcards
The role of DNA in heredity was first discovered by studying what?
bacteria and the viruses that infect them
Frederick Griffith
1928: worked with two strains of a bacterium, one pathogenic and one harmless
transformation
a change in genotype and phenotype due to assimilation of foreign DNA
Griffith’s experimental process
When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic
Oswald Avery, Maclyn McCarty, and Colin MacLeod
1944: announced that the transforming substance was DNA based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria
bacteriophages (or phages)
viruses that infect bacteria
Alfred Hershey and Martha Chase
1952: experiments showing that DNA is the genetic material of a phage known as T2
they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection
Erwin Chargaff
1950: DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group
DNA composition varies from one species to the next
Chargaff’s rules
that in any species there is an equal number of A and T bases, and an equal number of G and C bases
Maurice Wilkins and Rosalind Franklin
X-ray crystallography to study molecular structure
Franklin produced a picture of the DNA molecule using this technique
Franklin’s X-ray crystallographic images of DNA did what?
enabled Watson to deduce that DNA was helical
DNA shape and X-ray advantage
The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases
The width suggested that the DNA molecule was made up of two strands, forming a double helix
Franklin’s conclusion
two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
Watson and Crick
Determined pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray
adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) (Consistent with Chargaff’s rule)
Watson and Crick also suggested what?
that the specific base pairing suggested a possible copying mechanism for genetic material
Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication
Watson and Crick’s semiconservative model of replication
when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand
Matthew Meselson and Franklin Stahl
supported the semiconservative model
They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope
origins of replication
where the two DNA strands are separated, opening up a replication “bubble”
A eukaryotic chromosome may have hundreds or even thousands of origins of replication
Replication proceeds in both directions from each origin, until the entire molecule is copied
replication fork
At the end of each replication bubble is a Y-shaped region where new DNA strands are elongating
Helicases
enzymes that untwist the double helix at the replication forks
Single-strand binding protein
binds to and stabilizes single-stranded DNA until it can be used as a template
Topoisomerase
corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands
Where are nucleotides added to DNA elongation?
they can only add nucleotides to the 3’ end
primer
short RNA initial nucleotide strand for DNA elongation
primase
An enzyme that can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template
DNA polymerases
catalyze the elongation of new DNA at a replication fork
Most DNA polymerases require a primer and a DNA template strand
Rate of elongation
The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells
Where DNA begins elongation?
a new DNA strand can elongate only in the 5’ to 3’ direction
leading strand
where the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork
lagging strand
The opposite strand of the leading strand which is replicated
Elongation of the leading strand
synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase
DNA ligase
Joins the 3’ end of DNA that replaces primer to rest of leading strand and joins Okazaki fragments of lagging strand
“DNA replication machine”
The proteins that participate in DNA replication form a large complex
probably stationary during the replication process
Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules
DNA polymerases second function
proofread newly made DNA, replacing any incorrect nucleotides
Proofreading and Repairing DNA
In mismatch repair of DNA, repair enzymes correct errors in base pairing
DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example)
In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA
Replicating the Ends of DNA Molecules
The usual replication machinery provides no way to complete the 5’ ends, so repeated rounds of replication produce shorter DNA molecules
telomeres
Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences
Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules
telomerase
catalyzes the lengthening of telomeres in germ cells
If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce
Telomere potential focus
The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions
Proteins are the links between what?
genotype and phenotype
Gene expression
process by which DNA directs protein synthesis, includes two stages: transcription and translation
Archibald Garrod
1902: Suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions
He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme
George Beadle and Edward Tatum
exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media
one gene–one enzyme/protein/polypeptide hypothesis
Adrian Srb and Norman Horowitz
Identified three classes of arginine-deficient mutants
Each lacked a different enzyme necessary for synthesizing arginine
Point of RNA
RNA is the bridge between genes and protein synthesis
Transcription
the synthesis of RNA using information in DNA
Translation
the synthesis of a polypeptide, using information in the mRNA
Ribosomes
the sites of translation
Transcription and translation in prokaryotes.
translation of mRNA can begin before transcription has finished
Transcription and translation in eukaryotes.
the nuclear envelope separates transcription from translation
Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA
primary transcript
the initial RNA transcript from any gene prior to processing
The central dogma is the concept that cells are governed by a cellular chain of command: DNA → RNA → protein
The Genetic Code
20 amino acids, but there are only four nucleotide bases in DNA
triplet code
a series of nonoverlapping, three-nucleotide words
These words are then translated into a chain of amino acids, forming a polypeptide
the template strand
One of the two DNA strands provides a template for ordering the sequence of complementary nucleotides in an RNA transcript. The template strand is always the same strand for a given gene
However, further along the chromosome, the opposite strand may be the template strand for a different gene
codons reading order
the mRNA base triplets are read in the 5′ → 3′ direction
coding strand
the non-template strand because the nucleotides of this strand are identical to the codons, except that T is present in the DNA in place of U in the RNA
64 codons
deciphered by the mid-1960s
61 code for amino acids; 3 triplets are “stop” signals to end translation
Codons must be read in the correct reading frame (correct groupings) in order
Evolution of the Genetic Code
The genetic code is nearly universal, shared by the simplest bacteria and the most complex animals
Genes can be transcribed and translated after being transplanted from one species to another
the first stage of gene expression
Transcription
RNA synthesis is catalyzed by what?
RNA polymerase
RNA polymerase
pries the DNA strands apart and joins together the RNA nucleotides
does not need primer
promoter
The DNA sequence where RNA polymerase attaches
terminator
In bacteria, the sequence signaling the end of transcription
transcription unit
The stretch of DNA that is transcribed
The three stages of transcription:
Initiation
Elongation
Termination
Initiation
Promoters signal the transcription start point and usually extend several dozen nucleotide pairs upstream of the start point
Transcription factors
help guide the binding of RNA polymerase and the initiation of transcription
transcription initiation complex
The completed assembly of transcription factors and RNA polymerase II bound to a promoter
TATA box
promotor crucial in forming the initiation complex in eukaryotes
Elongation
As RNA polymerase moves along the DNA, it untwists the double helix, 10–20 nucleotides at a time
Nucleotides are added to the 3′ end of the growing RNA molecule
Transcription progresses at a rate of 40 nucleotides per second in eukaryotes
A gene can be transcribed simultaneously by several RNA polymerases
Termination in bacteria
the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification
Termination in eukaryotes
RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence
After transcription in eukaryotes
Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm
RNA processing
both ends of the primary transcript are altered
Also, in most cases, certain interior sections of the molecule are cut out and the remaining parts spliced together
Each end of a pre-mRNA molecule is modified in a particular way
The 5′ end receives a modified nucleotide 5′ cap
The 3′ end gets a poly-A tail
Purpose of pre-mRNA molecules caps
They seem to facilitate the export of mRNA to the cytoplasm
They protect mRNA from hydrolytic enzymes
They help ribosomes attach to the 5′ end
RNA splicing
Removing eukaryotic genes and their RNA transcripts that are long noncoding stretches of nucleotides that lie between coding regions
introns
The noncoding segments in a gene are called intervening sequences
exons
coding sections that are eventually expressed, usually translated into amino acid sequences
The removal of introns is accomplished by what?
Spliceosomes that consist of a variety of proteins and several small RNAs that recognize the splice sites
The RNAs of the spliceosome also catalyze the splicing reaction
Ribozymes
catalytic RNA molecules that function as enzymes and can splice RNA
Three properties of RNA that enable Ribozymes to function as an enzyme
It can form a three-dimensional structure because of its ability to base-pair with itself
Some bases in RNA contain functional groups that may participate in catalysis
RNA may hydrogen-bond with other nucleic acid molecules
alternative RNA splicing
Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing
domains
Proteins often have a modular architecture consisting of discrete regions
different exons code for the different domains in a protein
Exon shuffling
may result in the evolution of new proteins by mixing and matching exons between different genes
A cell translates an mRNA message into protein with the help of what?
transfer RNA (tRNA)
tRNAs transfer amino acids to the growing polypeptide in a ribosome
tRNA specificity
enables translation of a given mRNA codon into a certain amino acid
tRNA 2-D structure
Each carries a specific amino acid on one end
Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA
consists of a single RNA strand that is only about 80 nucleotides long
tRNA 3-D structure
twists and folds into a three-dimensional molecule
tRNA is roughly L-shaped with the 5′ and 3′ ends both located near one end of the structure
The protruding 3′ end acts as an attachment site for an amino acid
Accurate translation requires two instances of molecular recognition
First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase
Second: a correct match between the tRNA anticodon and an mRNA codon
wobble
Flexible pairing at the third base of a codon allows some tRNAs to bind to more than one codon
ribosomal RNAs (rRNAs)
two ribosomal subunits (large and small) are made of proteins
ribosome has three binding sites for tRNA
P site
A site
E site
P site
holds the tRNA that carries the growing polypeptide chain
A site
holds the tRNA that carries the next amino acid to be added to the chain
E site
the exit site, where discharged tRNAs leave the ribosome
The three stages of translation:
Initiation
Elongation
Termination
Translation Initiation
When the small ribosomal subunit binds with mRNA and a special initiator tRNA
The initiator tRNA carries the amino acid methionine
Then the small subunit moves along the mRNA until it reaches the start codon (AUG)
Proteins called initiation factors bring in the large subunit that completes the translation initiation complex
Translation Elongation
amino acids are added one by one to the C-terminus of the growing chain
Each addition involves proteins called elongation factors
Translation proceeds along the mRNA in a 5′ → 3′ direction
The ribosome and mRNA move relative to each other, codon by codon
Translation Termination
Elongation continues until a stop codon in the mRNA reaches the A site
The A site accepts a protein called a release factor
The release factor causes the addition of a water molecule instead of an amino acid
This reaction releases the polypeptide, and the translation assembly comes apart
Translation Elongation 3 Steps
codon recognition, peptide bond formation, and translocation
Energy expenditure occurs in the first and third steps
Empty tRNAs released from the E site return to the cytoplasm, where they will be reloaded with the appropriate amino acid
Often translation is not sufficient to make a functional protein so what happens?
Polypeptide chains are modified after translation or targeted to specific sites in the cell
Post-Translational Modifications
a polypeptide chain begins to coil and fold spontaneously into a specific shape: a three-dimensional molecule with secondary and tertiary structure
Two populations of ribosomes are evident in cells
Free ribosomes mostly synthesize proteins that function in the cytosol
Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell
Ribosomes are identical and can switch from free to bound
Polypeptide synthesis always begins where?
in the cytosol
Synthesis finishes in the cytosol unless what?
unless the polypeptide signals the ribosome to attach to the ER
signal peptide
Polypeptides destined for the ER or for secretion are marked by this.
It is a sequence of about 20 amino acids at or near the leading end of the polypeptide
signal-recognition particle (SRP)
binds to the signal peptide
The SRP escorts the ribosome to a receptor protein built into the ER membrane
The signal peptide is removed by an enzyme
polyribosome (or polysome)
Multiple ribosomes can translate a single mRNA simultaneously
enable a cell to make many copies of a polypeptide very quickly
coupling transcription and translation
bacterial cell streamlined process
newly made protein can quickly diffuse to its site of function
Mutations
changes in the genetic information of a cell
Point mutations
changes in just one nucleotide pair of a gene
change of a single nucleotide a DNA template strand
can lead to the production of an abnormal protein
Point mutations within a gene can be divided into two general categories:
Single nucleotide-pair substitutions
Nucleotide-pair insertions or deletions
nucleotide-pair substitution
replaces one nucleotide and its partner with another pair of nucleotides
Three types of nucleotide-pair substitution
Silent mutations
Missense mutations
Nonsense mutations
Silent mutations
have no effect on the amino acid produced by a codon because of redundancy in the genetic code
Missense mutations
still code for an amino acid, but not the correct amino acid
Nonsense mutations
Nonsense mutations change an amino acid codon into a stop codon; most lead to a nonfunctional protein
Insertions and deletions
additions or losses of nucleotide pairs in a gene
These mutations have a disastrous effect on the resulting protein more often than substitutions do
frameshift mutation
may alter the reading frame which could affect how the gene is expressed
Spontaneous mutations
can occur during errors in DNA replication or recombination
Mutagens
physical or chemical agents that can cause mutations
Chemical mutagens fall into a variety of categories
Most carcinogens (cancer-causing chemicals) are mutagens, and most mutagens are carcinogenic
CRISPR-Cas9
In bacteria, the protein Cas9 acts together with a guide RNA to help defend bacteria from viral infection
Cas9 protein will cut any sequence to which it is targeted
Scientists can introduce a Cas9–guide RNA complex into a cell they wish to alter
The guide RNA is engineered to target a gene
Cas9 cuts both strands of the targeted gene
The broken ends trigger a DNA repair system
The repair enzymes remove or add some random nucleotides while joining the broken ends
This is a way for researchers to “knock out” (disable) a given gene, to study what the gene does in an organism
They can introduce a template with a normal (functional) copy of the gene to be corrected
In this way, the CRISPR-Cas9 system edits the defective gene and corrects it
gene editing
altering genes in a specific way
