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the two strands of DNA separate and each serves as a template for the synthesis of a new strand.
Each new molecule has one old strand and one new strand.
Principles of DNA replication
- 2. Energy requirements are met by the use of dNTP forms of the monomers.
- The hydrolysis of the high energy phosphate bond provides the energy for the reaction.
- 3. Synthesis proceeds 5' to 3' by the addition of the new base onto the 3'-OH group of the sugar.
The enzyme that actually synthesizes DNA using a DNA template
DNA polymerase III alone carries out only two processes:
- 1. chain elongation- the addition of new nucleotides onto a 3'-OH group of a sugar on an existing strand in a template dependent manner.
- 2. Editing- the enzyme can proof read what it synthesized and if a mis-match in base pairing has occurred, it backs up to the mistake degrading the DNA, and resynthesizes it.
polymerase III therefore needs two macromolecules for it to synthesize DNA.
- 1. Template- this is the molecule to be used as a guide for the synthesis of a new strand following the rules of base pairing.
- 2. Primer- this is the molecule that provides the free 3'OH to which DNA polymerase III can attach the next base. This molecule must be already base paired to part of the template while the remainder of the template, the part to be used, is left single stranded.
Other DNA function requirements
- 1. Initiation of replication.
- 2. Separation of the DNA strands.
- 3. Unwinding of the DNA- twists build up as strands are separated if ends are not free. Ends are never free in circular or very long molecules.
- 4. Discontinuous synthesis- since DNA replication only occurs 5' to 3', and since the strands have opposite polarity, one strand has to be replicated going away from the replication fork.
Enzyme that separates the DNA strands
Single Strand Binding Protein or SSB
keeps strands separated
This protein binds to the already separated strands and keeps them from coming back together.
It is displaced by the replicating apparatus as it moves through.
The extra twisting or winding that accumulates in DNA as the helicase separates strands are eliminated by this enzyme
The enzyme that carries out this restart process in a discontinuous strand.
Makes an RNA molecule not a DNA molecule
The spacing between the regions where DNA primase makes RNA is about 1000 to 2000 nucleotides in E. coli, but much shorter in eukaryotes.
- To finish replication, a repair enzyme, DNA polymerase I, chews up the RNA and replaces it with DNA.
- This enzyme uses the 3'OH group on the newly synthesized DNA fragment to elongate. Finally, an enzyme called DNA ligase joins the two DNA fragments together.
Proof Reading function of DNA polymerase III involves two functions:
- 1. A 3' to 5' exonuclease- an activity that chews up DNA from a 3' end towards the 5' end.
- 2. Measuring mis-matches.
- Many of the enzymes involved in the replication process physically bind to each other at the replication fork to form this.
- This includes: DNA polymerase III, helicase, DNA primase, and perhaps other components.
- a unit of DNA that is replicated from a single initiation site
- This is not a unit in terms of the discontinuous strand, but of the double helix.
An enzyme that adds short, tandemly sequence repeated DNA sequences at the end eukaryotic chromosomes.
describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA via a rolling circle mechanism.
are polymers of amino acids strung together by peptide bonds.
The amino acids differ on the basis of their side chains, the R group.
- It is the 3D structure that gives a protein the ability to catalyze a specific reaction.
- Proteins catalyze reactions by bringing the substrates in contact, or by distorting bond angles to break them, or by providing groups at the site of the reaction that helps it go.
- All the DNA has to encode is the primary structure the protein, the amino acid sequence, in order to dictate the folding of the protein into its active state.
- There are three codons that call for no amino acids. TAA, TAG, TGA.
- These are stop codons that signal the cell machinery that it has come to the end of the coding sequence.
Mutations that generate one of these codons through a base pair substitution are called nonsense mutations.
- These lead to premature termination of the protein as we saw for some of the mutations in Yanofsky's experiments
- The start codon is almost always the methionine codon ATG
a change in a single base pair, substituting another base pair for it.
no amino acid substitution results from the base pair change,
an amino acid substitution
A missense mutation can be conservative, when a like amino acid is substituted which does not alter the overall structure, or therefore, possibly the function, of the protein.
- the substitution of a base pair that results in a change to one of the three nonsense codons
- Such mutations almost always cause a loss of protein function unless it occurs close to the end of the protein.
result from a small deletion or insertion
In order to transcribe a specific gene, there are signals in the DNA that encode:
- 1. start
- 2. stop
- 3. how often to transcribe
- 4. when to transcribe.
the process whereby internal RNA sequences are deleted from the RNA.
This is necessary because the protein coding sequence of many eukaryotic genes is interrupted by non-protein coding segments.
These segments must be deleted before the information can be read by the protein synthetic machinery of the cell.
- the process whereby the RNA molecule that carries the triplet code for the amino acids is used as a template for the synthesis of the protein.
- Translation requires an organelle, the ribosome, many smaller molecules, lots of energy.
DNA o RNA o protein
transcription The reaction requires:
- 1. Four ribonucleotide triphosphates: ATP, GTP, CTP, UTP.
- 2. A DNA template.
- 3. The enzyme DNA-dependent RNA polymerase
The core enzyme which is required for the enzymatic reaction consists of five protein subunits.
2 a, one each of b, and b' and w: a2bb'w
The core enzyme will synthesize RNA using a DNA template, but will not initiate transcription at the proper places (the beginning of genes) nor terminate properly (at the end of genes).
represents the signal required for the initiation of transcription, both in terms of where transcription starts and how frequently the gene will be transcribed (how many RNA copies will be made over a given period of time).
There are two types of termination signals in E. coli genes:
- p-dependent and p-independent.
- p is a protein that is required for p-dependent termination.
- p-dependent termination requires specific sequences in the DNA that results in the formation of a stem-loop structure in the RNA.D-independent termination also involves a stem-loop structure followed by a tract of U's
Eukaryotic DNA-dependent RNA polymerases
- 1. RNAP I transcribes RNA molecules that become part of the ribosome. These ribosomal RNAs, or rRNA, play a structural role and do not encode proteins.
- 2. RNAP II transcribes genes that encode proteins. This type of RNA is called the messenger RNA, or mRNA.
- 3. RNAP III transcribes the small RNAs such as transfer RNAs, or tRNAs, that play a role in protein synthesis.
Initiation by RNAP II in eukaryotes
- Initiation requires the DNA consensus sequence TATAAA located 30 bp upstream from the start of transcription.
- RNAP II plus a set of about a half-dozen proteins, called TATA-factors and designated TFIIA, TFIIB, etc, form a complex at this site.
- This complex is competent to transcribe very infrequently, i.e., transcription occurs only occasionally.
- High frequencies of transcription require additional proteins, called transcriptional activators are required to activate the TATA complex.
- These activators bind to specific DNA sequences that can be very far away, thousands of base pairs, from the TATA complex.
- They make contact with the TATA complex by looping the DNA between out.
5' and 3' modification
- As the RNA is being synthesized, the 5' end is modified by the addition of the Cap.
- The cap is a G residue linked to the free triphosphate at the 5' end of the RNA in a 5'-5' linkage.
- RNAs without this modification can not be used in protein synthesis. The 3' end of the RNA is modified by the addition of 100 to 200 A residues.
- These residues, referred to as the poly A tail, are added in a template independent reaction, i.e. these residues are not encoded in the DNA.
- The poly A tail is added by a poly A polymerase.
- This tail aids in translation and makes the RNA more stable in the cell.
Intervening sequences or Introns
These interrupting sequences
The splicing reaction is carried out in the nucleus by a complex structure containing both a number of proteins and small RNA molecules
The ribosome provides both:
- 1. the scaffolding on which the tRNA and mRNA are brought together,
- 2. and some of the enzymatic activity required for peptide bond formation
The bacterial ribosome is comprised of two subunits
- 30S subunit 16S rRNA - 1,500 nt
- 20 proteins
- 50S subunit 23S rRNA - 2,900 nt
- 5S rRNA -120 nt 34 proteins
- Protein synthesis can be divided into three stages, initiation, elongation, and termination.
- Initiation requires: the mRNA, the ribosome, a special tRNAFMET which is charged with formyl- methionine, three proteins IF-1, IF-2 and IF-3, and GTP
Initiation requires specific sequences on the mRNA
- 1. A methionine codon, AUG.
- 2. A Shine-Dalgarno sequence- a short (4-9 nt) sequence about 7 nt before (5' to) the AUG which are complementary to the 3' end of the 16s rRNA.
The steps of initiation are
- 1. The three initiation factors plus GTP bind to the 30S subunit.
- 2. The mRNA and the f-met-tNRAFMET bind to the 30S with the proper codon-anticodon pairing. IF-3 dissociates.
- 3. The 50S joins the complex causing GTP cleavage to GDP, and the dissociation of the GDP and IF-1 and IF-2.
The steps of elongation are as follows:
- 1. The amino acyl tRNA that is called for by the codon exposed in the A site (remember the fmet-tRNAMET is in the P site) enters the ribosome with the help of a protein called EF-Tu plus GTP. In the process of tRNA entering the A site, the GTP is hydrolyzed to GDP and EF-Tu leaves the ribosome.
- 2. The peptide bond is formed by the cleavage of the aminoacyl bond of the fmet- tRNAMET, and the formation of the peptide bond between the carboxyl terminus of the fmet and the amino terminus of the new amino acid on the tRNA in the A site.Thus, the direction of protein synthesis is amino terminus to carboxyl terminus.
- The enzymatic activity for peptide bond formation is called peptidyl transferase, but it is unclear which ribosomal protein carries this out or even if the rRNA is involved.
- 3. The next step is called translocation. The tRNA at the P site (which no longer has an amino acid attached) exits the ribosome, and the tRNA at the A site (now with the dipeptide) shifts to the P site. This movement also requires movement of the ribosome one codon down the mRNA to keep the codon-anticodon pairing. Translocation requires another protein, EF-G, plus GTP cleavage to GDP.
After translocation, the A site is empty and the cycle can repeat.