Translation Flashcards
What is translation?
Conversion of mRNA into a polypeptide chain
What is the genetic code?
the correspondence between the sequence of the 4 bases in mRNA & the 20 amino acids found in proteins
Features of the genetic code?
Triplet code
Non-overlapping
Degenerate
Universal
What are synonymous codons?
They all code for the same amino acid
What are tRNAs?
The adaptor molecule used for decoding the base sequence of mRNA into the amino acid sequence of proteins
Link between mRNA & the building blocks of proteins
Made by transcription
What is the primary structure of a tRNA molecule?
Small nucleic acids of 70-90nts
15 invariant & 8 semi-variant residues in tRNA
Have a 5’ monophosphate (rather than a 5’ triphosphate)
Contain modified bases – aren’t incorporated in the standard process of transcription, they come from the modifications of the standard bases in the post transcriptional process
What are the modified bases in the primary structure of tRNA molecules?
Ribothymidine (curly T)
Pseudouridine (fork)
Dihydrouridine (D)
Inosine (I)
What is the secondary structure of a tRNA molecule?
D loop – contains dihydrouridine
T loop – contains pseuouridine
Variable arm – 3 to 21 nucleotides
Anti-codon loop – mRNA interacts with the anticodon loop
Amino acid acceptor site
Base pairing in the stems but not in the loops
tRNA 3D structure arises from 9 extra hydrogen bonds
What is an aminoacyl tRNA?
A charged tRNA
A tRNA joined to an amino acid
How are aminoacl tRNAs formed?
This is a 2 step reaction driven by ATP
1) ATP is added to the carboxyl group of the amino acid to give a high energy intermediate called an aminoacyl adenylate
2) The aminoacyl adenylate reacts with the appropriate uncharged tRNA to give aminoacyl tRNA & AMP
Catalysed by tRNA synthetases
Are the tRNA synthetases all the same?
No
Some synthetases join the amino acid to the 2’ hydroxyl & some to the 3’ hydroxyl but once joined the 2 species can spontaneously interconvert
What is the process of joining amino acids to tRNA molecules?
Aminoacylation of tRNAs
Aminoacylation drives protein synthesis as the aminoacyl tRNA bond is of higher energy than the peptide bond
What are the 2 classes of aminoacyl tRNA synthetases and how are they different?
Class I
Class II
Differ in the way they recognise tRNA – has to recognise the right AA & also the right tRNA
The 2 classes bind different faces of the tRNA molecule
How do class I aminoacyl tRNA synthetases bind to the tRNA?
On the underside of the L-shape
How do aminoacyl tRNA synthetases proofread?
The acylation site rejects amino acids that are larger than the correct one
The editing site hydrolytic cleaves and rejects amino acids are smaller than correct one
DOUBLE SIEVE MECHANISM
Proofreading improves the fidelity of protein synthesis so that mistakes are made less than once every 10000 amino acids
What is the cognate tRNA for serine?
tRNA^Ser
What is the cognate aminoacyl tRNA synthesise (uncharged) of serine?
Seryl-tRNA synthetase
What is the charged aminoacyl tRNA of serine?
Seryl-tRNA^Ser
or
Ser-tRNA^Ser
How do codon-anticodon interactions work?
Codon-anticodon bases pair in an antiparallel fashion
Several features suggest something is missing
• 61 codons but only 20 tRNAs – some tRNAs recognise more than 1 codon
• Base at the 3’ end of the codon isn’t always important
What is the Wobble hypothesis by Francis Crick?
The first 2 bases of the codon base pair with the anticodon according to the usual base pairing rules
The base at the 5’ end of the anticodon can form non-standard hydrogen bonds with the base at the 3’ end of the codon
What are the 4 wobble base pairs?
I-C
I-U
I-A
G-U
How are ribosomes involved in translation?
Ribosomes are the particles on which protein synthesis (translation) takes place
They move along the mRNA & align successive amino acyl tRNAs & amino acids attached one by one to the growing polypeptide chain
Ribosomes are large RIBONUCLEOPRTEIN (RNA-protein) complexes
What is the composition of prokaryotic ribosomes?
The 70S structure is the complete prokaryotic ribosome
This can be divided into 2 sub-particles – 50S & 30S
What are the 3 stages of translation?
INITIATION
ELONGATION
TERMINATION
What does initiation require?
Requires initiation factors 1,2 & 3 and GTP (for energy)
The initiator tRNA fMet-tRNA^fMet recognises AUG at the initiator codon but does not recognise methionine codons when they are inside the open reading frame
• charged with N-formyl methionine
Anticodon more flexible so can recognise GUG as a start codon in E.coli
What does elongation require?
Requires elongation factors EF-Tu EF-Ts EF-G & GTP (energy)
What does termination require?
Requires a stop codon
Requires release factors RF1 or RF2
What happens in initiation?
1) The 30S subunit binds the ribosome binding site (RBS) by recognition of the Shine Dalgarno sequence
2) This allows the initiator tRNA (fMet-tRNAfMet) to bind to the start codon AUG
3) This allows the 50S subunit to bind, forming the 70S Initiation Complex on the mRNA, reading to begin translation
What are the 3 distinct sites within the ribosome?
The A site – for the acyl group when the amino-acyl tRNA binds
The P site – peptidyl where the tRNA attached to the peptide is located
The E site – where the uncharged tRNAs get pushed along to
How is the shine dalgarno sequence involved in translation?
The Shine Dalgarno sequence (mRNA) is complementary to the 3’ end of the 16S rRNA
Base pairing between the Shine Dalgarno sequence and the 16S rRNA properly positions the 30S ribosomal subunit on the mRNA
What is the role of initiation factors in the initiation of translation?
1) IF1 and IF3 bind to a free 30S subunit and their job is to get the 30S subunit on to the mRNA and to prevent the 50S subunit binding to it, unless it is first bound to a messenger RNA.
2) IF2 complexed with GTP binds the 30S subunit attaches to a mRNA.
3) A charged initiator tRNA can then bind and base pair with the AUG.
4) IF3 is displaced.
5) A 50S subunit can now bind which displaces IF1 and IF2 and GTP is hydrolysed in this energy consuming step, forming the complete 70S initiation complex.
How do elongation factors work?
1) The first step is the delivery of a charged amino acyl tRNA to the A site
2) There is peptide bond formation between the two tRNAs, during which the amino acid joined to the tRNA in the P site loses contact with its tRNA.
3) Then there is translocation where the mRNA is hauled through the ribosome so that the peptidyl tRNA is moved to the peptidyl site.
What does the delivery of the charged tRNA in elongation require?
EF-Tu, EF-Ts and GTP
EF-Tu is the factor that delivers the charged tRNA to the A site by forming a complex, requiring GTP hydrolysis.
However, there is now GDP stuck to the elongation factor. Therefore, EF-Ts recycle the EF-Tu back to the GTP form which can bind the next charged amino acid
What does peptide bond formation in elongation require?
Peptide bond formation is catalysed by the ribosome itself so doesn’t need any additional elongation factors
What does the translocation step in elongation require?
The translocation step requires EF-G and GTP
No recycling factor is needed in this case because GDP is released at the same time as the phosphate
How does termination occur?
Proteins called release factors interact with STOP codons
RF1 recognises UAA and UAG
RF2 recognises UAA and UGA
RF3.GTP (RF3 is bound to GTP) helps RF1 or RF2 carry out termination
RRF (ribosome release/re-cycling factor) & EF-G promote dissociation of the ribosome from mRNA
What do release factors do?
The release factors direct the ribosome so that the peptide bond is cleaved from the peptidyl tRNA so the polypeptide chain is released. The uncharged tRNA is removed and the ribosome dissociates off the mRNA with the help of RRF, EF-G and GTP hydrolysis
