Lecture 1: Mechanism of Protein Synthesis Flashcards
How are tRNAs charged?
Charging is the addition of an aminoacyl by an aminoacyl tRNA synthetase.
• The synthetase recognises the amino acid and the tRNA.
• Amino acid is added to the enzyme with ATP. This forms enzyme-amino acid-AMP and pyrophosphate.
• This is then added to tRNA to form amino acid tRNA, AMP and the enzyme.
How are amino acids recognised?
Recognition involves non-covalent interactions between amino acids and enzymes.
• The difference between some AAs is very small. A methylene group contributes very little binding energy. It would only discriminate to a level of 1 in 100.
• The recognition system therefore needs 2 independent discrimination events.
• These steps must be separated by an irreversible (energy requiring) step in order to be multiplicative.
• There is a double sieve. The first sieve involves size and steric requirements, the second involves chemical characteristics.
What is the structure of the 70S ribosome?
The 70S ribosome is made up of the 50S and 30S subunits.
• Both ribosomes are 60 : 40 RNA to protein.
• 50S contains 23 rRNAs and 5S RNA.
• 30S contains 16S rRNA.
• There are 3 sites: A, P and E between the 2 subunits.
• RNA domains fit together to make a single huge RNA mass that determines the 50S structure. The acidic globular domains of the proteins fill the gaps and cracks between the RNA on the surface of the 50S subunit.
• The proteins are evenly distributed over the surface of the ribosome except for the active site cleft and the 30S interaction face where they are absent.
• Positively charged tails on 12 of the proteins (gly and pro) make extensive specific interactions with the RNA helices. This stabilises the structure.
What is the evidence for the A and P sites on the ribosome?
• Take a cell free system containing ribosomes, buffer (with magnesium ions, 14C-phenylalanine-tRNA, ATP/GTP) and varying concentrations of soluble cell extract.
• This will yield ribosome carrying polyPhe-tRNA. Not free polyPhe-tRNA or poly Phe-tRNA unbound to ribosomes.
• Ribosomes are washed to remove soluble proteins and ATP/GTP.
• They are then incubated with puromycin. Puromycin is an aa-tRNA analogue.
• About 50% of ribosomes release 14C labelled polyPhe as polyphe-PM, where the carboxyl of the polyPhe has made a bond to the amino group of PM. PM can’t bind to the ribosome so it’s released.
• This shows that the ribosome catalyses the formation of the peptide bound. This also shows that additional energy supplying material isn’t required.
There is a second part of the experiment.
• We wash the ribosomes to remove the PM.
• Add soluble cell extract and GTP then more PM. It is observed that the remaining 50% of the ribosomes release 14C-Phe that is now transferred to give Phe-PM.
• This shows that the ribosomes have a PM-reactive site and another PM unreactive site for carrying peptidyl tRNA This also shows that soluble cell extract can translocate form PM-unreactive to PM-reactive site.
• PM can be used to distinguish the 2 sites on the ribosome. The ribosome binding site for peptidyl tRNA that reacts with PM is the P site. The other site that can carry aminoacyl tRNA is called the A site.
• These other factors involved in protein synthesis are factors such as EFTu.
• EFTu binds to GTP and the aa-tRNA. It binds all correctly charged aa-tRNAs with higher affinity.
What is mischarging? How does it happen and how can we measure it?
Mischarging is when an amino acid is put on the wrong tRNA.
• Mutations could involve the anticodon, D loop or acceptor stem. This suggests that the synthetase inspects the whole conformation as well as the details of base recognition.
• Modified nucleotides imply that shape is important too.
• We can measure this by measuring the incorporation of an amino acid into a protein that doesn’t encode it. E.g. cysteine in flagellin.
• Frequency is 1 in 10,000.
• The two recognition steps in protein synthesis must have an error frequency no worse than this.
What is the structure of tRNA?
- They have similar secondary structures (stems and loops).
- They also have similar tertiary structures: anticodons, D-loops, acceptors and T-arm stacking.
- Conserved nucleotides maintain the shape. There must also be differences to distinguish sequences and amino acids.
- The first anticodon base is in the wobble position. This is because some anticodons can pair with more than one codon due to wobble base pairing.
- Coaxial stacking is when two RNA duplexes form a continuous helix. This is stabilised by base stacking. The interacts lead to the functional L-shaped tertiary structure.
- rRNAs and tRNAs are heavily base stacked and show triple base interactions in which a base forms an interaction in the minor groove of a Watson and Crick base pair.
