Lecture 16 - Translation Flashcards
What is the universal genetic code?
Universal genetic code
There are 61 amino acid coding codons and three stop codons
* Has some degeneracy: all amino acids except Met & Trp are encoded by more than one codon.
* AUG used in the initiation of protein synthesis.
Redundancy in the third position only
What types of RNA are involved in translation?
mRNA - Encodes protein
rRNA - Part of ribosome used to translate mRNA into the protein
tRNA - Couples the region of ribosome which binds the mRNA codon and amino acid.
Describe the structure of tRNA?
tRNA has a simple cloverleaf structure. It has a:
* Anticodon loop
* D-loop
* T-loop
* CCA tail - added post transcriptionally. It is the site of attachment of amino acids
tRNA contains several modified bases. (Pseudouridine and Dihydrouridine)
Has hairpin loops
Describe tRNA attachment to its cognate amino acid.
Each tRNA species must be loaded with the amino acid that corresponds to the mRNA codon to which the tRNA binds. This amino acid is refered to as the cognate.
The process is called aminoacylation or charging
It is carried out by aminoacyl-tRNA synthetases using ATP as a cofactor
Produces an aminoacyl-tRNA (or charged tRNA).
Aminoacyl-tRNA synthetases use various chamical features, such as charge, hydorphobicity, size and shape to descriminate between the different amino acids.
- The aminoacylation reaction - The amino acid is attached to the 3’ or 2’ OH group of the 3’ terminal adenine nucleotide of the tRNA.
- There are at least 20 different enzymes - one for each amino acid - absolutely specific for tRNA an amino acid.
○ Size exclusion - amino acid to big and can’t fit in aminoacylation site
○ Editing pre-transfer - can fit so binds with AMP however tRNA is not added and it instead moves to second active site (editing site) and is recognised as the incorrect amino acid and it dissociates
○ Editing post-transfer - Can fit so bind with AMP and binds to tRNA but is then taken to the editing site and it dissociates
Correct amino acid - Enters acylation site binds to AMP and then correct tRNA (as correct cant enter editing site) and leaves the enzyme as aminoacyl tRNA
Describe codon-anticodon pairings.
Codon-anticodon pairings can be based on normal Watson-Crick interactions.
However for the binding between the first base of the anticodon and the third base of the codon other types of base pairing are allowed (wobbles).
Wobbles provide part of the basis for the degeneracy of the genetic code
3’ G or U can bind to two different codons
If binds to I can bind to three different codons
What is the amino acid normally first to be incorporated.
With rare exception, methionine is the first amino acid incorporated into the proteins. Although it may be removed later.
A specific tRNA molecule, tRNAfmet (prokaryotes) or tRNAimet is always used at the start and another for elongation involving methionine ( tRNAmmet)
What is the small and large ribosomal subunit.
rRNA comprises a large part of the ribosome - the diagrams above show the secondary structure and 3D organisation of the small (L) and large (R) ribosomal subunits.
In bacterial ribosomes the large subunit is 50S and the small is 30S together = 70S
In eukaryotic ribosomes the large is 60S and the small is 40S together = 80S
Describe translation initiation in prokaryotic cells.
The Ribosome has three sites.
A - Amino-acyl site
P - Peptidyl site
E - Exit site
- The Shine-Dalgarno sequence (consensus sequence) on the mRNA is recognised by the anti-shine-Dalgarno on the 16S rRNA.
- The initiator f-Met-tRNA is guided to the P site of the ribosome with the help of three initiation factors (IF1, IF2 and IF3)
- IF1 (amino-acyl) and IF3 (exit) independently associate with the small ribosomal subunit in the regions where the A and E site tRNAs usually bind, leaving the P site unfilled. Therefore they direct the initiator tRNA to the P-site. They also prevent premature joining of the small and large ribosomal subunit. The GTPase IF2 also helps with this.
- Once the initiator tRNA has been deposited in the P site, the large subunit joins the small subunit so that elongation can begin. The GTPase IF2 couples the energy of GTP hydrolysis to the joining of the subunit leading to the displacement of all three initiation factors.
Describe the decoding step of translation elongation.
During the decoding step the ribosome selects and aminoacyl-tRNA with an anticodon that is complementary to the mRNA codon in the A site.
This aminoacyl-tRNA is bound to the elongation factor EFTu-GTP (eEF1A-GTP in eukaryotes) which interacts with the ribosome to ensure high fidelity in the selection process.
Before the different phases of the selection process: initial selection and proofreading.
The selection of a cognate aminoacyl-tRNA in the A site of the ribosome involves two distinct steps separated by the hydrolysis of GTP by EFTu. In the “initial selection” phase, an aminoacyl-tRNA complexed with EFTu can be rejected from the ribosome before EFTu hydrolyses GTP. Following GTP hydrolysis and the departure of EFTu, ribosomes still can reject near-cognate tRNA in a “proofreading” phase. Additionally, cognate tRNAs promote more rapid GTPase activation and accommodation, such that kinetic discrimination mechanisms also contribute to the overall fidelity of tRNA selection.
Describe peptide bond formatio nin translation elongation.
After a cognate aminoacyl-tRNA has been accommodated in the A site of the ribosome, the aminoacyl- and peptidyl tRNAs are ready for the catalysis of peptide bond formation (the growing peptide chain is referred to aminoacyl-tRNA).
The formation of a peptide bond between amino acids is catalysed on the ribosome by the peptidyl transferase centre.
The growing polypeptide chain is transferred from the Site to the A site tRNA. At some point following peptidyl transfer, the tRNAs are thought to assume a hybrid state of tRNA binding in preparation for translocation—we depict this hybrid state as a translocation of the tRNAs relative to the large subunit, but not with respect to the small subunit.
The peptidyl transferase reaction is NOT catalysed by a normal protein enzymes
It is carried out by a RNA section of the 50S subunit known as a Ribozyme
Ribozymes are involved in a number of cellular processes including the splicing for RNA molecules and the removal of introns from mRNA
What is translocation?
After peptide bond formation the hybrid state is formed. Next, the anticodon end of the tRNA (still base-paired with the codon) moves with respect to the small subunit in a reaction catalysed by EFG (eEF2).
The ribosome moves one codon along in the 3’ direction. This requires EFG and GTP. Peptidyl RNA moves from the A site to the P site. Uncharged tRNA moves from P site to E site.
EFG is then released (required GTP hydrolysis), Can be reused.
EFG is a structural mimic of EFTu:tRNA.
It binds to the ribosome competitively with EFTu (binds at the A site). GPT hydrolysis is coupled to conformational changes in EFG and ribosome.
The altered conformation forces movement of the peptidyl-tRNA from the A site to the P site and pushes the deacylated tRNA into the exit site.
The ribosome therefore moves three bases in the 3’ direction on mRNA
Describe elongation and termination during translocation.
Elongation
Repeated steps of tRNA binding, peptidyl transferase and translocation
Insertion of aminoacyl-tRNA at A site dislodges the uncharged tRNA in the E site.
The peptide hain gest longer and longer: up to 100 amino acids or more.
Termination
When one of the three stop codons is reached, there is no tRNA available to enter the A site, instead a Release Factor (RF) binds to the stop codon
* RF1 recognises stop codons UAA and UAG
RF2 recognises stop codons UAA and UGA
What is ribosome recycling.
Ribosome recycling factor (RRF) and EFG-GTP promote complex disassembly. IF3 binds to the small subunit to stabilise it in its dissociated state.
