aminoacylation

Question 1

how are amino acids addded to tRNA?

Answer 1

aminoacylation

Question 2

what does Aminoacyl - tRNA synthetases do?

Answer 2

catalyzes the attachment of amino acids to the correct tRNA. MUST have binding pockets for the proper tRNA, the proper amino acid and ATP

Question 3

what does AlaRS do?

Answer 3

adds alanine to tRNA

Question 4

what does PheRS do?

Answer 4

adds phenylalanine to tRNA

Question 5

what does TrpRS do?

Answer 5

adds tryptophan to tRNA

Question 6

how many steps is aminoacylation and which step requires ATP?

Answer 6

2 step process and ATP is required in the 1st step

Question 7

what type of reaction is aminoacylation?

Answer 7

SN2 displacement reaction

Question 8

why is it a 2 step reaction?

Answer 8

there is no leaving group so one must be added

Question 9

describe the steps of aminoacylation;

Answer 9

1st the amino acid is activated by attachment of AMP
This releases pyrophosphate and energy
The activated aminoacyl adenylate remains bound to the enzyme for the 2nd step
2nd aminoacyl-tRNA synthetases catalyze a nucleophilic attack from the OH group on the ribose of the terminal adenosine on the tRNA 3’ CCA tail to the carbonyl C of the amino acid
aminoacyl-tRNA synthetases carries out both steps of the reaction

Question 10

how do class 1 and class 2 aminoacyl-tRNA synthetases differ?

Answer 10

Class 1 = 2’ Oh attack, monomeric
Class 2 = 3’ OH attack, dimeric

Question 11

what bond is susceptible to hydrolysis?

Answer 11

The high energy bond between the amino-acid and the tRNA

Question 12

how is the amino acid - tRNA bond protected from spontaneous hydrolysis?

Answer 12

by the immediate binding of charged tRNA with the elongation factors EF-Tu (bacteria) eEF1A (eukaryotes)

Question 13

what are the characteristics of identity elements?

Answer 13

May include both sequence and structural features
Located primarily in the anticodon loop and/or acceptor stem
The 2 classes use different element

Question 14

describe the tRNA^Ala element and its base pair that is recognized;

Answer 14

G3:U70 in the acceptor stem. As long as the base pair is GU, alanine will be added to the acceptor stem by AlaRS

Question 15

what is a cognate amino acid?

Answer 15

The correct amino acid fro a tRNA

Question 16

what is a cognate tRNA?

Answer 16

The correct tRNA for an amino acid

Question 17

what are the 2 sites in aminoacyl-tRNA synthetase?

Answer 17

aminoacylation and editing

Question 18

what is the first quirk of aminoacyl-tRNA synthetase?

Answer 18

blocks anything that is too large or doesn’t fit properly, but cannot block smaller, similar side chains

Question 19

what is the 2nd quirk of aminoacyl-tRNA synthetase?

Answer 19

hydrolyses the amino acid if it is too small

Question 20

can isoleucine fit in the ValRS active site?

Answer 20

no

Question 21

can Val fit in the IleRS active site?

Answer 21

yes

Question 22

does Ile fit in the IleRS editing site?

Answer 22

no

Question 23

can Val fit in the IleRS editing site?

Answer 23

yes

Question 24

describe editing pre transfer;

Answer 24

Can be checked at either the activation or aminoacylation step - some check the identity of the aminoacyl adenylate and if the amino acid is incorrect it can be edited before being added to tRNA

Question 25

describe editing post transfer;

Answer 25

Can also check the aminoacylated tRNA to make sure that the correct amino acid has been added. If incorrect it can be edited

Question 26

what did the raney nickel experiment show?

Answer 26

that translational machinery cannot distinguish correct vs incorrect

Question 27

what is transamidation?

Answer 27

Some bacteria and archaea have fewer than 20 aa-tRNA synthetases often the enzymes for attaching glutamine and asparagine to their cognate tRNAs
In this case, glutamic acid is first added to tRNA^gln bu GluRS
A transamidase then changes the side chain of the attached amino acid from an acid to an amide, producing glutamine bound tRNAs
The transamidase recognizes the amino acid only when it is attached to the tRNA
GluRS must be able to recognize 2 different tRNAs and the transamidase must be able to distinguish between them.

Question 28

what is cysteine desulfurase?

Answer 28

CysRS is missing in methanococcus jannaschii. In that case a phosphorylated serine is loaded by a special enzyme — SepRS
Cysteine desulfurase converts the attached serine to cysteine

Question 29

what is the initiator tRNA?

Answer 29

Initiator tRNA^met is only used for initiation
The initiation apparatus of protein synthesis must recognize this tRNA and no any other
The general protein synthesis apparatus must NOT recognize this
The initiator codon = AUG
Eukaryotes - tRNAi^Met
Bacteria - tRNA^fMET
Formyl methionine is used in bacteria and is added by transformylase. It must distinguish between the initiator and the regular
In bacteria: MetRs must be able to recognize 2 different tRNAs and transformylase must distinguish between them

Question 30

during protein synthesis what brings charged tRNAs to the ribosome but don’t bind with initiator tRNAs?

Answer 30

ancillary elongation factors eEF1A and EFTu

Question 31

what are the structural anti-determinants on initiator tRNAs that ensure they don’t bind to ancillary elongation factors eEF1A and EFTu?

Answer 31

The bacterial initiator has a C-A wobble mismatch
The eukaryotic initiator has an A-U pair
Both have 3 G-C pairs in the anticodon stem