Translation Flashcards
What are some features of the genetic code?
Degenerate
Non-overlapping
Triplet code
Almost universal (not ciliated protozoa)
4 nucleotide bases
3 bases per amino acid (1 codon) = 64 possibilities
How are codon triplets read?
Sequentially, from a fixed point
This was discovered due to studying mutations: deletion/insertion and therefore frame shifts
How was the genetic code determined?
Using polynucleotide phosphorylase - this makes poly-RNA without a template
Leading to cell free translation systems
Adding certain bases lead to the discovery of certain bases e.g. Adding Poly(A) leads to synthesis of Poly(Lys) - AAA = lysine
Nitrocellulose filter binding assay - this tested the ability of trinucleotides with known sequence to promote binding of specific amino-acyl tRNAs to ribosomes = identification of codons
What are some specific codons?
Codons that produce the same amino acid = synonyms
Most synonyms differ only in the 3rd base
Start codons = AUG and GUG (met and val)
Stop codons = UAG, UAA and UGA
Describe the ribosome?
Ribosomes - small organelles acting as molecular machines that make proteins
Predominantly made up of RNA
Binds to mRNA so the codons can be read with high fidelity
It catalyses the formation of the peptide bond
It has 3 tRNA binding sites: A, P & E - aminoacyl site, peptidyl site and exit site
Describe the prokaryotic ribosome?
2 subunits
Large subunit = 50S = (5S & 23S rRNA + 31 proteins)
Small subunit = 30S = (16S rRNA + 21 proteins)
Total = 70S
Proteins not evenly distributed
Few proteins are near the sites at which tRNA & mRNA are bound
RNA-protein bridges are mediated by Mg2+
Describe prokaryotic rRNA?
All 3 rRNA is contained in one operon, but it is extensively processed into complex secondary structures
Describe the eukaryotic ribosome?
2 subunits
Large subunit = 60S = (5S, 5.8S & 28S rRNA + 49 proteins)
Small subunit = 40S = (18S rRNA + 33 proteins)
Total = 80S
They are larger than prokaryotes
Describe eukaryotic rRNA?
There are several hundred tandemly repeated copies of rRNA genes
It has a buried prokaryotic ribosome
They have expansion segments (not homologous to prokaryotes) and additional proteins on the solvent exposed surface
Small nucleolar RNA’s (snoRNA) direct methylation of rRNA
What are some facts of translation?
Polypeptide synthesis proceeds from the N-terminus to the C-terminus
Chain elongation occurs by linking the growing polypeptide to the incoming tRNA’s amino acid residue
Ribosomes read mRNA in the 5′→3′ direction
Active translation occurs on polysomes (polyribosomes)
What is the N-terminal residue of a polypeptide in prokaryotic translation?
N-formylmethionine
It already contains an amide bond, which allows it to be the first residue in the polypeptide chain
How is a tranlation initiation site also determined in prokaryotic translation, other than an initiation codon?
Shine-Dalgarno sequence:
A purine rich tract of 3-10 nt and 10 nt upstream from the start codon
Base-pairing interactions between an mRNA’s Shine–Dalgarno sequence and the 16S rRNA permit the ribosome to select the proper initiation codon
Describe initiation P1 of prokaryotic translation?
IF-3 binds to the 30S subunit of the inactive ribosome - preventing the reassociation of 50S subunit
IF-1.GTP captures mRNA to the 30S subunit
IF-2 brings tRNA-fMet to the 30S subunit
IF-1 - blocks the aminoacyl site (A-site)
IF-2 - is bound to the N-formylmethionine tRNA and binds to the peptidyl site (P-site)
IF-3 - blocks the exit site (E-site)
The Shine-Dalgarno sequence positions the mRNA
Describe initiation P2 of prokaryotic translation?
IF-3 is released from the E-site and the 50S subunit binds
This causes IF-2 to hydrolyse GTP -> GDP
Therefore IF-1.GDP is released from the A-site
This reaction rearranged the 30S subunit and releaes IF-2
= 70S initiation complex
Describe elongation of prokaryotic translation?
Decoding
EF-Tu & GTP bound to an aa-tRNA, whose anticodon is complementary to the mRNA codon, brings the molecule to the A-site
GTP -> GDP allowing EF-Tu & GDP to be released
Transpeptidation
The amino acids form a peptide bond via peptidyl transfer
(meanwhile EF-Ts reloads the EF-Tu with GTP, GTP will then displace EF-Ts)
Translocation
EF-G & GTP binds to the ribosome and GTP -> GDP
This drives the translocation of the tRNA molecules from P & A sites to E & P sites (Order: E P A)
This continues until a stop codon is reached
How does the ribosome monitor the correct codon-anticodon pairing in prokaryotic translation?
The first codon–anticodon base pair: is stabilised by the binding of rRNA A1493 base (in the bp minor groove)
The second codon–anticodon base pair: is stabilised by the binding of A1492 and G530 (in the bp minor groove)
The third codon–anticodon base pair: is stabilised by the binding of G530 (in the bp sminor groove)
This is a more flexible interaction to consider wobble pairs of non-Watson-Crick bp
What allows the ribosome to carry out proof reading within elongation of prokaryotic translation?
GTP hydrolysis by EF-Tu is a thermodynamic prerequisite, to allow ribosomal proofreading to take place
If EF-Tu hydrolyses GTP then aa-tRNA assumes a more energetically favourable position as a result - proving the correct aa was chosen
How is the peptide bond formed in prokaryotic translation within elongation?
It is formed through the nucleophilic attack of the amino group, of the 3′ aa–tRNA in the A site, on the carbonyl group of an ester to form a tetrahedral intermediate that collapses to an amide and an alcohol
This doesn’t need ATP due to the ester bond between the polypeptide and the P-site tRNA is a ‘high energy’ bond
The peptidly transferase centre (catalysing the peptide bond formation) is located on the 50S subunit, mainly rRNA
Ribosome = Ribozyme
It catalyses the reaction by properly positioning and orienting the substrates and/or excluding water from the active site
How is translocation of prokaryotic elongation stabilised?
When the P site tRNA moves to the E-site and the A-site tRNA moves to the P-site
The mRNA moves with them, to act as a placekeeper to preserve the reading frame
An Mg2+ stabilsed kink in the mRNA, between A and P codons, prevents slipping
The sites use negative allosteric cooperativity, with different affinities for different tRNA molecules at different stages of the cycle
Describe termination P1 of prokaryotic translation?
RF-1 (or RF-2) occupies the A-site when a stop codon reaches the A-site
GGQ motif (present in RF-1) helps cleave the completed polypeptide chain - causing peptidyl transfer to water, leaving a uncharged tRNA and a free polypeptide that dissociates from the ribosome
RF-3 & GTP bind in the A-site causing the release of RF-1 (or RF-2)
GTP hydrolysis to GDP causes the release of RF-3
Describe termination P2 of prokaryotic translation?
RRF - ribosomal recycling factor binds in the A-site followed by EF-G & GTP
EF-G hydrolyses the GTP, causing RRF to move to the P-site and the tRNAs occupying the E and P sites to be released
The large and small subunits of the ribosome dissociate, facilitated by the binding of IF-3
RRF, EF-G and mRNA are released
What is a nonsense suppressor tRNA?
They recognise nonsense mutations that have created a stop codon premature to the end of a translation sequence
It prevents chain termination
Give an overview of eukaryotic initiation?
2 types: cap dependent and cap independent
Most use cap dependent, whilst eukaryotic viruses use independent
43S pre-initiation complex = 40S + itRNA and eIF
48S complex = 43 complex + mRNA
80S complex = 40S + 60S + mRNA + itRNA
Describe initiation P1 of eukaryotic translation?
eIF3, eIF1A and eIF1 bind to the 40S subunit
• eIF1 - binds to the E-site of 40S subunit + helps recruit eIF2
• eIF1A - prevents premature binding of tRNA to A-site
eIF2 & GTP recruit methionine-tRNA to 40S subunit
eIF5B & GTP bind to eIF1A, which will eventually ensure the dissociation of all eIF
= 43S pre-initiation complex
