aminoacyl-tRNA syntheses Flashcards
what are the two steps of AAtRS action?
activation of the amino acid
charging of the tRNA
what happens during activation of the amino acid?
the amino acid is activated as a high energy bond is formed with AMP, pyrophosphate is cleaved out by pyrophosphatase
what occurs during charging of the tRNA?
tRNA picks up the amino acid from the enzyme bound adenylate, AMP is released alongside 2x Pi
which parts of the tRNA do the enzymes sample?
acceptor stem, anticodon loop, editing site
where do class I aa-tRS enzymes add the amino acid?
onto the 2’ OH of the adenosine of the 3’ CCA terminal
where do class II aa-tRS enzymes add the amino acid?
onto the 3’ OH of the adenosine of the 3’ CCA terminal
where does esterification between enzyme and amino acid take place?
between OH of ribose and carboxyl of amino acid
how does the aaRS recognise amino acids?
via the R group
what problem arises from aa-RS recognition of cognate amino acids?
amino acids that are smaller than the cognate amino acid are able to enter the active site
what can happen if an amino acid that is not cognate for the aaRS is picked up?
there is no way of preventing false activation of the amino acid, but after this:
1 - production of a non-cognate product
or
2- hydrolytic editing of the falsely activated intermediate
why is hydrolytic editing ‘editing at a price’?
still uses cleavage of pyrophosphate to generate AMP, but no product is generated, energetically expensive
what is the error frequency of false activation?
1 in 200
what is the error frequency of hydrolytic editing?
1 in 800
how do we know that activation and hydrolytic editing are two separate steps?
one step must be irreversible, this is the cleavage of PPi by pyrophosphatase
what is the total error frequency of activation and charging of aa-tRNA?
1 in 160,000
what is meant by the double sieve model?
the idea that there are 2 active sites on the aaRS, the amino acid is added to the activation site, where the tRNA is now floppy enough to transfer activated amino acids into the editing site, where non-cognate aas will be edited out
what do elongation factors do?
bind to amino-acyl-tRNA, which then can bind to the ribosome
how is elongation factor Tu a mechanism of ribosomal proofreading?
it has a tryptophan that provides a signal when GTP is bound vs when GDP is bound
GTP - high affinity for the ribosome
GDP - low affinity for the ribosome
aa-tRNA-Tu-GTP binds too the A site on codon-anticodon complex
what does elongation factor Ts do?
it is an exchange protein which displaces guanine
how does the stoichiometry of GTPase provide a measure of proofreading?
if there is greater than 1 GTP/ aa-tRNA selected, this indicates proofreading is occurring
what must ribosomes do?
generate a water free zone to enable codon-anticodon pairing
monitor codon-anticodon pairs using decoding site
