Protein synthesis: translation Flashcards
General components needed for protein synthesis (7)
-mRNA
-tRNA
-ribosomes
-energy carriers: ATP/GTP
-amino acids
-aminoacyl-tRNA synthetases
-protein synthesis factors (initiation, elongation and termination factors)
Characteristics of the genetic code
-triplet code
-degenerate
-comma less (arrangement of triplet codons on mRNA is one after another without a gap)
-non-overlapping
-universal
-unambiguous (each codon specifies ONE amino acid)
Crick and Brenner experiments (1961)
AIM: determine general features of genetic code
PROCESS:
-T1 bacteriophages treated with with the proflavine mutagen (which causes insertion/deletion of a single DNA base)
CONCLUSION: code in non-overlapping, triplet and degenerate
describe the structure of tRNA
-small RNA molecules (150 nucleotides long)
-CCA sequence on 3’ due to the maturation process
4 MAIN DOMAINS:
1. Amino acid attachment site - at CCA terminus
2. Anticodon loop: region which recognises and binds to complementary codon on mRNA
3. DHU loop (left)
4. TpsiU loop (right)
-contains a small ‘extra arm’ between TpsiU and anticodon region that is VARIABLE
-structure is maintained by weak Hbonds
What is the starting codon?
AUG which codes for methionine
what are the 3 stop codons?
UAA UAG UGA
How many possible amino acid codons are there in the mRNA
61
REASON: 64 total minus the stop codons (not methionine though)
How many molecules of tRNA are there that carry anticodons?
Around 35-45
REASON: although there are 61 codons that specify for an amino acid, there are less than 61 tRNA molecules because in anticodon-codon recognition, the third nucleotide has imperfect/weak pairing
Due to this occasional non-canonical pairing, a single molecule of tRNA can recognize more than one codon through its anticodon loop
Does the ribosome have proofreading activity in the case that an incorrect amino acid is carried by the tRNA moleule?
NOOO!!
!! this is why the action of enzymes (aa-tRNA transferases) that attach amino acid onto tRNA molecule must be very accurate
What is the role and structure of aa-tRNA transferase
ROLE: perfectly recognised tRNA and corresponding amino acid and link them together
STRUCTURE:
-ATP binding site
-amino acid site
-tRNA site
how many aa-tRNA transferases do we have
20 - one for each amino acid
mode of action of aa-tRNA transferases
- enzyme binds an amino acid and ATP molecule
- activated amino acid by adenylating it (adding AMP) which also released a pyrophosphate
- interacting of tRNA with the now charged aa
- release of AMP and aa is covalently bonded to the 3’ CCA site of the tRNA molecule
SOS: tRNA is now charged whereas before aa asociation it was neutral
What feature of the aa-tRNA transferase allows high fidelity of the process?
Problem faced: in the amino acid site of the transferase, aa larger than intended would not fit, but aa smaller than intended might fit –> causes error
Solution: PROOFREADING: secondary editing pocket which would be smaller than main pocket and would allow the smaller aa to fit and form associations. Then the aa would be hydrolysed.
!!! editing pocket needs to be smaller than main pocket so the target aa doesn’t fit inside
Structure of ribosomes
-major and minor subunit that are assembled to form a fully active ribosome
-can either be associated with RER or be free in cytosol
Prokaryotes: 70S ribosomes, 50S major and 30S minor
Eukaryotes: 80S ribosomes, 60S major and 40S minor
what are polysomes
2 or more ribosomes associated with an mRNA molecule
what is a ribozyme?
an RNA molecule capable of acting as an enzyme
what is different about the path of the very first tRNA arriving at the ribosome?
The first tRNA (bringing methionine) enters the P instead of the A site –> allows the second tRNA to get into the A site and the formation of the first peptide bond
What is the assembled organisation of a ribosome?
-rRNA responsible for catalytic activity
-ribosomal proteins that maintain stability and integrity
-mRNA binding groove located on small subunit
CREATION OF 3 SITES:
1. A site: allows charges tRNA entry
2. P site: holds tRNA while connected to the growing peptide chain
3. E(exit) site: where uncharged tRNA (after giving up aa) stays until leaving ribosome
Initiation in prokaryotes
-30S subunit starts dissociated from its larger unit and associated with the initiation factors: IF1 & IF3
-IF3 goes into E site to prevent ribosome from forming again
-IF1 goes into A site making only P site available for 1st tRNA
-H bonds form between 16S protein of small subunit and the Shine-Dalgarno sequence of mRNA(5’) - this permits the AUG codon to be perfectly placed in the P site
-IF2 bound to tRNA allows it to also recognised SD sequence and bind in P site to form initiation complex
-IF2 is GTP bound and upon correct tRNA position in P site it hydrolyses GTP to GDP which allows IF1/2/3 to leave complex
-this allows the large subunit to be able to associate and form full ribosome
ways in which initiation differs in prok and euk
- euks don’t have Shine Dalgarno sequence, and instead the initiation factors recognise the 5’ mRNA cap
- IF4 recognises the cap (not IF2) and the complex runs along mRNA until the start codon is reached and recognised
- Codzag sequence - specific consensus sequence which the AUG codon must be in to be considered the start codon
3 general steps of elongation
- aa-tRNA entering into the A site
- peptide bond formation
- translocation
elongation process - step 1
- tRNA enters A site by associating with an elongation factor: EF-Tu which is linked to a GTP
INVOLVES 2 CONTROL CHECKPOINTS:
1) EF-Tu GTP factor:
-in the case of perfect codon/anticodon pairing the GTP is hydrolysed to GDP and EF-Tu loses affinity for ribosome (causing its dissociation)
-In the case of imperfect pairing the GTP is not hydrolysed and the tRNA is released
2) Accommodation:
- tRNA is twisted and only the tRNAs that have strongly bound anticodons and codons will resist the twist.
-badly-paired tRNAs will be released
elongation step 3
TRANSLOCATION
-after peptide bond formation (larger subunit mediated) everything moves 3 nucleotides
-This uses the EF-G-GTP elongation factor) which hydrolyses GTP to GDP, causing a conformational change that makes EF-G look similar to EF-Tu
-EF-G can hence enter into A site and push all components by a codon
Termination process
Triggered by stop codons bcos there are no tRNA anticodons for the stop codons
Termination factors enter A site and cause release of protein
Dissociation of ribosome into 2 subunits
