What is Translation?
- protein ( polypeptide) synthesis
- nucelotide sequence information in DNA copied ( transcribed) into mRNA
- the mRNA like strand of the DNA is the coding strand
- the information in the mRNA is translated into the amino acid sequence of the corresponding polypeptide
How is translation carried out ?
-by the ribosome
( mRNA is read 5’ to 3’ direction)
- protein is synthesised from N- to C- terminus
What is the genetic code?
- specifies which mRNA nucleotide sequence corresponds to which amino acid in a protein
- the genetic code consists of non- overlapping triplets which are read from a fixed starting point ( francis crick)
what are the bases?
- AGCT(U)
how many different amino acids are there ? and therefore how many different varieties of polypeptides are there?
- there are 20 amino acids
- 1 base per amino acid - 4 possible code words
- 2 base per amino acid - 16 possible code words
- 3 base per amino acid - 64 possible code words
Cracking the genetic code?
- evidence against overlap or bracketing of code words ( codon)
- single base changes in genes = single amino acid changes in protein
- frameshift mutations affect the whole downstream sequence (i.e. no gaps /bracketing )
- there was evidence that triplet (3base) codons form from synthetic mRNAs translated in vitro.
- the code is uniersal- with a few excceptions where ther are limited cchanges ( the genetic code is identical in all organisms)
- main exception = mitochondria
what is triplet binding assay?
it tetsts which synthetic trinucleotides can promote binding of specific tRNAs to ribosomes
- tRNAs mediate the interaction of amino acids with the genetic code.
What is tRNA?
- transfer RNA
- it acts as an adapter molecultes
- processed from longer precursor RNAs
- it bridges the gap between codon and amino acid
- each tRNA has a triplet anticodon that recognises one orr more codons in the mRNA by base pairing
- tRNAs bring the correct amino acid to the ribosome in response to a specific codon
- the amino acid is added to the growing polypeptide,
HAS UNIVERSAL ‘ CLOVERLEAF ‘ STRUCTURE - defined by 4 stems and 4 loops ( the stem and leaf are called an arm)
- base pairs differ - depending on tRNA
What is the tRNA structure?
- secondary structure : - maintained by invariant base pairs
- tertiary structure:- L-shaped ; maintained by tertiary hydrogen bonds between the invariant bases
tRNAs need specific features (e.g. for charging enzymes)
tRNAs need common features - e.g. all bind to same site on ribosomes.
What do we know about tRNA base modifications?
tRNA’s are the most heavily modifieed post transcriptiional RNA
- tRNAs have may post - transcriptionally modified bases.
What is the role of tRNA base modifications?
- to make the binding of different aminoacyl-tRNAs to both the A and P sites of the ribosome uniform
- without the mpdifications - the ttRNA sequence and the particular amino acid linked to the tRNA affect the binding affinity for the two principal tRNA binding sites on the ribosome
What is anticodon modifications?
- modified bases in the anticodon can affect the nature and efficiency off base pairing with codons.
what is meant by tRNA charging ?
- covalent linkage of the tRNA 3-end to the cognate( means as specified by the anticodon) amino acid
Catalysed by an aminoacyl-tRNA synthetase
- one enzyme per amino acid
- each recogniises all isoaccepting tRNAs
- linkage is via an ester bond to the 3’ - OH group
What is the two step - mechanism of tRNA charging reactions?
alpha alpha + ATP»_space; aa ~ AMP + PPi
2.
alpha alpha~AMP =tRNA»_space; aa ~ tRNA + AMP
( refer to slide 20 to get a better understanding !)
Why is accuracy of charging critical?
- mischarged tRNAs lead to errors in proteins
How is accuracy mantained ?
- it is controlled by the aminoacetyl - tRNA synthetases
- each recognises all itss isoaccepting tRNAs by conntacts on the acceptor stem and anticodon loop
- contacts aligned on one face of the tRNA
- usually at least one anti-codon base is recognised
- single base changes in the acceptor stem change the tRNAs identity
- isoaccepting tRNA molecules contain an invariant discriminator ( present in all tRNAs to encode amino acids)base in the acceptor stem that controls their identity
Speed versus accuracy ?
proofreading as a mechanism to avoid trading off one for the other !
- both amino acid selection and tRNA selection are subjecct to proofreading (1.e. error checking mechanisms )
- proofreading either difavours the forward reaction ( kinetic proofreading) or reverses the catalytic reaction ( chemical proofreading) is the wrong component has been selected.
- avoids the need for absolute accuracy (slow)
another way that accuracy is maintianed is amino acid selection- what do you know about that?
Amino acid selection:
- chemical proofreading occurs on binding the tRNA - either the incorrect aminoacyl adenylate is hydrolysed or tRNA mischarging occurs followed by hydrolysis.
(e.g. Ile and Val are very similar)
What do the active site and editing site have in relation to maintaining accuracy?
- active site = excludes too large amino acids
- editing site- can accept ones that are too small!
active and editing site form double molecular sieve
- conformational change in the acceptor stem of the tRNA moves the amino acid from the catalytic site to the editing site.
What is proofreading and why is it important ?
- tRNA selection - kinetic proofreading
- correct tRNA binding induces a rapid conformational change - stabilising binding to allow fast aminoacylation
- incorrect tRNA fails to induce the conformational change - allows tRNA time to dissociate before being mischarged,
- the right tRNA produces rapid translation - the wrong one does not -so gives time to sort it !
- mutation affects the accuracy of the enzyme - the enzyme has normal level of expression, activity , solubility and normal structure.
- mutation is in editing site of enzyme
- defect is proofreading - leads to higher levels of mis-charging with serine.( results in mix up of serine and alanine )
- the experiment with a mouse ( causing neurodegenerative condition) shoes the need for quality control of tRnA charging !
What is decoding?
- refers to codon- anticodon recognition ( occurs on ribosome)
- by base pairing between the codon and anticodon
- ‘wobble’ and modified bases allow one tRNA to decode more than one codon
What is the wobble hypothesis?
this is an extension to the normal rules of base pairing !
- watson - crick base pair rules between anticcodon position 1 and codon position 3 are relaxed due to flexibilty of the anticodon loop !
- wobble rules explain how a single tRNA can decode a codon pair
- also shows that unique codons must end in G or U because A and C cannot have a unique meaning !
- modified bases restrict or enhance base- pairing in the wobble position.
What is a summary of Decoding?
- multiple tRNAs can recognise the same codons
- single tRNAs can recognise multple codons
- Wobble rules define a minimal set of 31 tRNAs plus an initiator tRNA for translating the universal genetic code.
( a special tRNA is required for protein synthesis)
