aminoacyl-tRNA syntheses

Question 1

what are the two steps of AAtRS action?

Answer 1

activation of the amino acid

charging of the tRNA

Question 2

what happens during activation of the amino acid?

Answer 2

the amino acid is activated as a high energy bond is formed with AMP, pyrophosphate is cleaved out by pyrophosphatase

Question 3

what occurs during charging of the tRNA?

Answer 3

tRNA picks up the amino acid from the enzyme bound adenylate, AMP is released alongside 2x Pi

Question 4

which parts of the tRNA do the enzymes sample?

Answer 4

acceptor stem, anticodon loop, editing site

Question 5

where do class I aa-tRS enzymes add the amino acid?

Answer 5

onto the 2’ OH of the adenosine of the 3’ CCA terminal

Question 6

where do class II aa-tRS enzymes add the amino acid?

Answer 6

onto the 3’ OH of the adenosine of the 3’ CCA terminal

Question 7

where does esterification between enzyme and amino acid take place?

Answer 7

between OH of ribose and carboxyl of amino acid

Question 8

how does the aaRS recognise amino acids?

Answer 8

via the R group

Question 9

what problem arises from aa-RS recognition of cognate amino acids?

Answer 9

amino acids that are smaller than the cognate amino acid are able to enter the active site

Question 10

what can happen if an amino acid that is not cognate for the aaRS is picked up?

Answer 10

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

Question 11

why is hydrolytic editing ‘editing at a price’?

Answer 11

still uses cleavage of pyrophosphate to generate AMP, but no product is generated, energetically expensive

Question 12

what is the error frequency of false activation?

Answer 12

1 in 200

Question 13

what is the error frequency of hydrolytic editing?

Answer 13

1 in 800

Question 14

how do we know that activation and hydrolytic editing are two separate steps?

Answer 14

one step must be irreversible, this is the cleavage of PPi by pyrophosphatase

Question 15

what is the total error frequency of activation and charging of aa-tRNA?

Answer 15

1 in 160,000

Question 16

what is meant by the double sieve model?

Answer 16

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

Question 17

what do elongation factors do?

Answer 17

bind to amino-acyl-tRNA, which then can bind to the ribosome

Question 18

how is elongation factor Tu a mechanism of ribosomal proofreading?

Answer 18

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

Question 19

what does elongation factor Ts do?

Answer 19

it is an exchange protein which displaces guanine

Question 20

how does the stoichiometry of GTPase provide a measure of proofreading?

Answer 20

if there is greater than 1 GTP/ aa-tRNA selected, this indicates proofreading is occurring

Question 21

what must ribosomes do?

Answer 21

generate a water free zone to enable codon-anticodon pairing

monitor codon-anticodon pairs using decoding site