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Molecular Genetics 2
Transcript of Molecular Genetics 2
"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... "Semi-Conservative" 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 ("origins") on a chromosome DNA Polymerase: Enzyme Responsible for DNA synthesis. Can only add deoxyribonucleotides to the 3' end.
Helicase: Opens the helix (which causes strand separation)
Single Strand Binding Proteins ("SSBP's"): Keep the strand open
Primase: Puts down a small RNA primer which is necessary for DNA polymerase to bind to at the origin.
Topoisomerase: Rotates the DNA to decrease torque (which would shred the helix.
DNA replication can only occur in the 5' to 3' direction.
DNA is "anti-parallel": 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 "Replisome" 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. "Proof-Reading" Telomeres: The ends of eukaryotic chromosomes.
Consist of a short, repeating DNA sequence
Vertebrate Telomere: TTAGGG Telomerase: The enzyme responsible for replicating the ends of eukaryotic chromosomes.
Uses an RNA template to add more telomere sequence during replication.
Not active in senescent cells. 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 "proof-reading" 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
Heritability DNA RNA How transcription happens: 1. Initiation RNA polymerase attaches to a "promoter" region in front ("upstream") 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 "TATA Box" 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:
Rho-independent: the transcript bases hydrogen bond with themselves, fold back and pull the transcript out of RNA polymerase.
Rho-dependent: 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 (mRNA): Carries DNA sequence information to the ribosome
Transfer RNA (tRNA): Carries specific amino acids to the ribosome
Ribosomal RNA (rRNA): 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 ("introns") interspersed between coding DNA ("exons") 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 spliceosome (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") RNA polypeptide The Genetic Code: The Ribosome tRNA 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 the an amino acid attached is said to be "charged" Triplet code: mRNA is read in units of three bases ("codons")
There are 64 possible codons (for 20 possible amino acids).
The code is redundant and unambiguous.
The code has "start" and "stop" 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.
Methionine is brought to the start codon (AUG) by the methionine tRNA.
The ribosome assembles so that the start codon (AUG) 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 ("Translocates"). 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 (UAG, UAA, or UGA) 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 "anti-codon" loop in the tRNA molecule tRNA "anti-codon" Protein Synthesis in Total Prokaryote & Eukaryote
Translation Specifics Prokaryotes Since prokaryotes do not have a nucleus, transcription and translation can be coupled.
Polyribosomes: simultaneous translation of a transcript (even while that transcript is still being made. Eukaryotes 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. 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 Silent The substitution changes a codon to another codon for the same amino acid. Missense The substitution changes a codon to a codon for a different amino acid. Nonsense 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 indel's occur in multiples of three. PS: 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 need to be made at the endoplasmic reticulum. Recruits an SRP protein, which modulates "docking" of the ribosome to the rough ER. An RNA "hairpin" 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 replication, 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 Transcription Translation Mutation Blame it on the DNA Sickle Cell Again! The Genetic Code Splicing