Transcription is the process by which the polymerisation of ribonucleotides guided by complementary base pairing produces an RNA transcript for a gene
What are the 3 distinct phases of transcription?
What are the three post translational modifications?
Describe the initiation phase of transcription
- RNA polymerase recognises and binds to promoter regions; specialised DNA at the start of a gene where transcription will start
- The TATA box (the "core promoter region" of genes 25 base pairs upstream of transcription start site) binds transcription factors which in turn attracts RNA polymerase to start mRNA production
- After binding to the promoter region, RNA polymerase unwinds part of the double helix exposing unpaired bases on the template strand
Describe the elongation phase of transcription
RNA polymerase travels along the template strand adding nucleotides to the growing RNA polymer in the 5' to 3' direction
Describe the termination phase of transcription
RNA sequences that signal the end of transcription are known as terminators. There are two types of terminators:
1) Intrinsic terminators - Cause RNA polymerase core enzymes to terminate transcription on their own
2) Extrinsic terminators - require proteins other than RNA polymerase, particularly Rho, to bring about termination
Describe capping (post transcriptional modification)
A methyl-guanine cap is added to the 5' end following removal of a phosphate group by phosphatases. The methyl-guanine is bonded with a 5' - 5' triphosphate linkage to stabilise the mRNA
Describe polyadenylation (post transcriptional modification)
This involves cleavage at the 3' (tail) of the pre-mRNA which is then replaced by a series of adenine (A polyA tail)
Describe splicing (post transcriptional modification)
The removal of introns (regions of RNA that do not code for proteins) by endonucleases (please within the polynucleotide and exonuclease (cleave the ends of the polynucleotide) The end product is mature mRNA
Describe the initiation phase of translation
-At the 5' cap of the mRNA the 40s subunit of ribsome with Met-tRNA binds
- For translation to start, a starting codon must be recognised. This is 5' AUG which is a specific codon only for methionine
- The anticodon for this (found on the tRNA is 5'CAU
- The 60S subunit then binds for elongation to occur
Describe the elongation phase of translation
The RNA has two sites for tRNA to bind
1) The P site is for the site holding the peptide chain
2) The A site is for the site accepting the tRNA
- Met-tRNA occupies the P site and another aminoacyl-tRNA enters the ribosome to occupy the A site (requires GTP)
- The methionine forms a peptide bond with the next aminoacyl-tRNA making the P site now uncharged. This allows the ribosome to move along (translocation) The binding of the two amino acids requires PEPTIDYL TRANSFERASE
Describe the termination phase of translation
The termination of a polypeptide chain forming requires a stop codon to be read on the mRNA, Stop codons can be 5'UUA, 5'UAG or 5'UGA - there are no tRNAs that can bind to these codons and so the peptide and tRNA are hydrolysed to release the protein into the cytoplasm following the binding of relate factor
Explain why the triplet code is said to be degenerative?
There is more than one code per amino acid (64 codes for 20 amino acids)
Explain how mutations outside the coding region can affect gene expression
Mutations to promoter regions where transcription factors bind can affect gene expression, to either constitutively activate or deactivate it
What is the product of transcription?
Where does transcription occur?
What is the ORF region?
Open reading frame - area of the gene holding the code for all amino acids residues of the gene product
Name the key enzyme involved in translation
Peptide transferase - catalyses formation of peptide bonds between amino acids
List the 9 differences in prokaryotic protein synthesis compared to eukaryotes
1) Simpler promoter sequences
2) One type of RNA polymerase
3) No splicing or post transcriptional modification
4) Coupled transcription and translation
5) Short lived mRNA
6) Different transcription factors
7) Different translation factors
8) Distinctive translation initiation mechanism
9) Simpler ribosomes (70s-50s and 30s)
How can the differences in bacterial gene expression be exploited clinically?
Attacking the 30s and 50s subunits only which are unique to bacteria as eukaryotes have 60s and 40s sub units
What is rifampicin's mechanisms against bacteria?
Inhibits transcription by binding to RNA polymerase
What is methotrexates mechanism against bacteria?
Inhibits dyhydrofolate reductase which prevents the synthesis of of tetrahydrofolate from folic acid so DNA cannot be made
What is penicillins mechanism against bacteria?
Inhibits transpeptidase so cross links in bacterial cell walls cannot form resulting in lysis from the osmotic pressure
What is tetracyclines mechanism against bacteria
Inhibits translation by competing with tRNA at A site of bacterial ribosome
How can bacteria develop resistance to drugs?
- Increased efflux e.g. up regulation of P-glycoprotein
- Decreased influx
- Increase transcription of target
- High rate of division
- Altered target
What is the most common type of RNA found in the eukaryote cells?
rRNA - more than 80%
Why are there so many different types of mRNA in comparison to rRNA and tRNA?
mRNA is specific for each gene and different copies can be made from each gene including pre and mature mRNA
How can mutations outside the coding region affect gene expression?
Mutations can occur in promoter sequences and splice sites