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DNA Replication and Transcription/Translation Notes

Chapter 16 and 17 Notes

Jaclyn Severn

on 26 January 2017

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Transcript of DNA Replication and Transcription/Translation Notes

Universal across all domains of life.
The Central Dogma
Once the structure of DNA was determined, it was time to figure out how it worked

Central Dogma
": Term coined by Francis Crick to explain how information flows in cells.

Allows for Gene Expression
It is easy to see how DNA is copied by looking at it's structure
"It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material,"
-End of Watson & Crick's 1953 paper
But it still needed to be proven...
" Replication
Meselson & Stahl
3 Possible Hypotheses
"The most beautiful experiment in Biology"
Conducted an experiment in 1958 that supported the
semi-conservative model of replication
How Replication Happens:
1. Initiation
Replication can only begin at specific locations ("
") on a chromosome
DNA Polymerase
: Enzyme Responsible for DNA synthesis.

: Opens the helix (which causes strand separation)

Single Strand Binding Proteins ("
"): Keep the strand open

: Puts down a small RNA
which is necessary for DNA polymerase to bind to at the origin.

: Rotates the DNA to decrease torque (which would shred the helix.
The addition of new nucleotides into a strand of DNA is

Nucleotides are automatically added to the 3' end of the DNA strand, as determined by the sequence of the nucleotides in the opposite strand.

This means that DNA replication can only occur in the
5' to 3'

DNA is "
": Both strands have opposite 5' to 3' orientations (one is "upside-down" compared to the other)
2. Elongation
Nucleotides are added to the new strand of DNA in the 5' to 3' direction.

There is an issue: DNA is anti-parallel

As the replication machinery moves along the chromosome, only one strand of DNA (the "
leading strand
") can be made in a continuous, 5' to 3' piece.
The other strand (the "
lagging strand
") has to be made in smaller, discontinuous 5' to 3' segments ("
Okazaki fragments
") which are then stitched together by the enzyme ligase.
Leading Strand
Lagging Strand
The "
Replication Fork
The "
" Holoenzyme
Once it begins, replication proceeds in two directions from the origin
Prokaryotes vs. Eukaryotes
1 origin vs. Many Origins
The "
Replication Bubble
3. Termination
Elongation continues until replication bubbles merge.

The ends of linear eukaryotic chromosomes pose a unique challenge.

Each round of replication shortens the 5' end of the lagging strand (by about 100-200 bp).

If this continued indefinitely, chromosomes would get shorter and shorter after each replication. Information would start to be lost.
The ends of eukaryotic chromosomes.
Consist of a short, repeating DNA sequence

Vertebrate Telomere: TTAGGG
The enzyme responsible for replicating the ends of eukaryotic chromosomes.

Uses an RNA template to add more telomere sequence during replication.

Not active in
There are 5 different DNA polymerases described in prokaryotic cells. Eukaryotic cells have ~15.

They serve a variety of functions, well beyond the scope of this course.

We'll focus on one polymerase:

DNA polymerase III
- Is responsible for elongation. Rate of elongation is ~500 bases/second in E. coli

The eukaryotic analog DNA polymerase elongates at a rate of ~50 bases/second.

The initial error rate during elongation is 1 in 10,000 mismatched bases.

This would create 300,000 mutations every time a human cell divided.

There are a series of other DNA polymerases and nucleases responsible for "
" DNA.

Proof-reading reduces the error rate to 1 in 10 billion nucleotides during replication (less than 1 per 3 human cell divisions).

Proof-reading is a continual process.
Allows for
How transcription happens:
1. Initiation
RNA polymerase attaches to a "
" region in front ("
") of a gene
prokaryotes: RNA polymerase binds directly to the promoter.
eukaryotes: RNA polymerase requires an assemblage of "
Transcription factor
" proteins" to be able to bind to the promoter.

Promoters have characteristic DNA sequences (ex "
" in eukaryotes)
2. Elongation
Similar to DNA replication, RNA production occurs in a 5' to 3' direction.

The "
template strand
" of DNA is the one that the RNA transcript is being produced off of (its sequence is opposite to the transcript)

The "
nontemplate strand
" or "
coding strand
" of the DNA will have the same sequence as the RNA transcript (with thymines replaced by uracils in the transcript)
3. Termination
Transcript production continues until the end of the transcription unit is reached.

There are multiple mechanisms of termination, two examples from prokaryotes:
: the transcript bases hydrogen bond with themselves, fold back and pull the transcript out of RNA polymerase.

: The Rho protein destabilizes the RNA-DNA hydrogen bonding at RNA polymerase and ceases transcription.
What Happens Next?
Many Kinds of RNA
Unlike DNA, RNA plays many roles in the cell
There are ~10 described types of RNA, each with different functions, but there are three major types:

Messenger RNA (
): Carries DNA sequence information to the ribosome
Transfer RNA (
): Carries specific amino acids to the ribosome
Ribosomal RNA (
): Major structural building block of ribosomes

We'll just focus mRNA for now, since it has the information that will become protein. In prokaryotes, the mRNA transcript is immediately translated.

In eukaryotes, the mRNA transcript is extensively processed in the nucleus before it leaves to be translated.
Post-Transcriptional mRNA Processing
(Eukaryotes ONLY!)
5'Cap and poly-A tail
Exon Splicing
A modified nucleotide is added to the 5' end of the transcript.

A tail of several hundred adenine residues is put on the 3' end of the transcript.

These modifications function in nuclear export and maintenance of the mRNA
Eukaryotic genes contain large stretches of non-coding DNA ("
") interspersed between coding DNA ("
To produce a functional protein, the introns must be removed and the exons must be spliced together prior to the movement of the mRNA transcript to the nucleus.

This process is accomplished by a
(a type of enzymatic RNA molecule)
Why Introns?
It's a great question. Not really answered.

Evolutionary baggage? Selfish genes?

We do know that having multiple exons in a gene allows eukaryotes to make multiple functional proteins from one gene ("
alternative splicing
The Genetic Code:
The Ribosome
The site of protein synthesis.

The only "non-membrane" bound organelle.

All cells have ribosomes.

Composed of two subunits.

Has three "sites":
A site
: "Aminoacyl"- where amino acids enter the ribosome
P site
: "peptidyl"- where the growing polypeptide is kept.
E site
: "exit"- where empty tRNA molecules leave.
Transfer RNA molecules.

Responsible for bringing amino acids to the ribosome
Amino acids are added to tRNA molecules through the action of "
amino-acyl tRNA synthase
" enzymes.

A tRNA with an amino acid attached is said to be "
Triplet code: mRNA is read in units of three bases ("

There are 64 possible codons (for 20 possible amino acids).

The code is redundant and unambiguous.

The code has "
" and "
" punctuation.
pig with GFP from a jellyfish
tobacco plant with luciferase from a firefly
How translation happens:
1. Initiation
The mRNA attaches to the small ribosomal subunit.

is brought to the start codon (AUG) by the methionine tRNA.

The ribosome assembles so that the start codon (
) is in the P-site.

This is called the "
translation initiation complex
2. Elongation
The next codon determines the next amino acid to be brought to the ribosome.
The incoming charged tRNA enters at the A-site.
The growing polypeptide is transferred to the new tRNA molecule. A
peptide bond
is formed.
The ribosome shifts ("
"). The tRNA with the polypeptide is now in the P-site.

The uncharged amino acid is now in the E-site.
The next codon is now available in the A-site for the next incoming charged tRNA
3. Termination
When a stop codon (
, or
) is encountered, a release factor binds to the A-site.

The polypeptide chain is released.

The ribosome disassembles.
tRNA binding at the ribosome is mediated by an "
" loop in the tRNA molecule
tRNA "anti-codon"
Protein Synthesis in Total
Prokaryote & Eukaryote
Translation Specifics
Since prokaryotes do not have a nucleus, transcription and translation can be coupled.

: simultaneous translation of a transcript (even while that transcript is still being made.
Eukaryotes can not couple transcription and translation. They do not have polyribosomes.

Eukaryotes also need to target different polypeptides to different areas of the cell.
What is a gene?
The relationship between genes and proteins has been well established since the early 1900's.
George Beadle and Edward Tatum:
One Gene-One Enzyme
" hypothesis
Nobel Prize: 1958

Experiments looking at nutritional mutants in Neurospora fungus. Able to describe metabolic pathways and identify enzymes responsible.
What we know now
One gene-One enzyme: too simplistic.

Not all genes are for enzymes.
Not all genes are for protein.
Not all genes are for polypeptides.

Still, it's a good place to start.
Mutation Revisited
It becomes clear how changes in DNA can affect changes in protein structure, and in physiology. There are 2 major types of DNA-level mutations:

Point mutations
: One DNA base is replaced by another DNA base.
Frame-shift mutations
: DNA bases are inserted or deleted ("in/dels").

Each type of mutation can have different effects, depending on the situation.
Point Mutations
The substitution changes a codon to another codon for the same amino acid.
The substitution changes a codon to a codon for a different amino acid.
The substitution changes a codon to a stop codon
Frameshift Mutations
Extensive missense
The reading frame of the ribosome is altered so that all amino acids downstream from the in/del are altered
Immediate nonsense
The reading frame of the ribosome is altered so that a stop codon is introduced prematurely
Limited Effect
The reading frame is restored when insertions or deletions occur in multiples of three.
The code was cracked largely by Marshall Nirenberg

Put synthetic RNA into "cell free" E. coli extract and analyzed the polypeptides that were made.

Nobel Prize: 1968
Big Questions
Make Sure You Can

Signal Peptide
: a small signal peptide sequence on polypeptides that needs to be made at the endoplasmic reticulum. Recruits an SRP protein, which modulates "docking" of the ribosome to the rough ER.
This deer is albino, because of a genetic defect
An RNA "
" loop, similar to what happens in Rho-independent termination
Sickle Cell Anemia is due to a point mutation.
How is the structure of DNA related to its function?

How does DNA allow for heritability?

How does DNA allow for traits in an organism?

How do mutations affect DNA structure and function?
Explain all steps of replication, transcription and translation, the enzymes required for each and the flow of information from DNA to RNA to protein.

Compare repliacation, transcription and translation in prokaryotic and eukaryotic biological systems.

Interpret the genetic code and use it to determine the amino acid sequence of a polypeptide if given the DNA sequence.

Explain the relationship between DNA sequence, protein sequence, and phenotype of an organism.

Describe the possible effects of DNA-level mutations on protein structure and organismal phenotype.
DNA Replication
Blame it on the DNA
Sickle Cell Again!
The Genetic Code
Full transcript