Protein Metabolism I

Question 1

aminoacyl-tRNA synthetases

Answer 1

responsible for attaching amino acids to the 3’ CCA end of a tRNA

Question 2

Stop Codons

Answer 2

UAA, UAG, UGA

Question 3

Wobble Hypothesis

Answer 3

Canonical base pairing w/1st two codon bases (normal base rules); non canonical base pairing is possible at third site (non-normal base pairing);

Question 4

Why is the wobble effect hypothesis important?

Answer 4

1) kinetic advantage - faster protein synthesis 2) tRNA can dissociate more readily from the RNA template.

Question 5

How do aminoacyl-tRNA synthetases apply the correct amino acid?

Answer 5

They check for base pairing within’ the tRNA, and they also analyze modified bases.

Question 6

Aminoacyl tRNA synthetase Reaction

Answer 6

Goal: form an ester linkage between AA and tRNA. 1) charged with ATP forming AA-AMP intermediate. 2) Aminoacyl-AMP + tRNA goes to Aminoacyl-tRNA + AMP

Question 7

Class I aminoacyl-tRNA synthetases

Answer 7

1) Accommodate larger, more hydrophobic amino acid substrates; 2) enzymes are monomers

Question 8

Class II aminoacyl-tRNA synthetases

Answer 8

1) Accommodate smaller, more hydrophillic amino acid substrates 2) enzymes are dimers or multimers.

Question 9

Step 2 difference between class I and class II aminoacyl-tRNA synthetases

Answer 9

1) class I aminoacyl-tRNA synthetases transfer AA to the 2’OH first then to the 3’ OH via a transesterification reaction. 2) class II transfers AA directly to 3’OH

Question 10

Aminoacyl-tRNA synthetase sites

Answer 10

site 1: catalytic domain (interacts with the tRNA 3’end to load AA) ; site 2: variable domain ( interacts with the specific bases on the tRNA that identify the tRNA)

Question 11

Specificity of aminoacyl-tRNA synthetases to tRNA

Answer 11

1) one or more bases in anticodon 2) one or more bases in the acceptor stem 3) discriminator base 73

Question 12

Where does high fidelity of tRNA synthetases come from?

Answer 12

1) Size exclusion 2) editing pre-transfer 3) editing post-transfer.

Question 13

Editing pre-transfer

Answer 13

aminoacyl-tRNA synthetase accomodates activated amino acid; if rejected pre-transfer, then aminoacyl-adenylate is hydrolyzed

Question 14

Editing post-transfer

Answer 14

amino-acyl tRNA synthetase; if rejection occurs post transfer AA is cleaved from tRNA

Question 15

Dbp5

Answer 15

helicase; uses atp; pulls mRNA transcripts out of the nuclear pore

Question 16

Because prokaryotic mRNA is not capped, how are they recognized by ribosomes?

Answer 16

1) 3’ end of 16srRNA is recognized by purine rich shine-dalgarno sequence

Question 17

Initiation of transcription in Eukaryotes

Answer 17

1) Ribosome binds to 5’ cap with the aid of proteins IRES 2) Ribosome scans for AUG

Question 18

Pre-rRNA transcript

Answer 18

1) DNA segment that leads to the production of 30S subunit, 50S subunit; and the tRNA in prokaryotes. 2) methylation of pre-rRNA transcript 3) methylation guides proteins to cut transcript.

Question 19

Puromycin

Answer 19

antibiotic; binds to the ribosomal catalytic site blocking protein synthesis.

Question 20

GAPs

Answer 20

GTPase- activating protein; promotes the hydrolysis pf GTP by EFTu once aminoacyl-tRNA is in the A site

Question 21

GEF

Answer 21

guanine-nucleotide exchange factors; recharge GDP to GTP

Question 22

Prokaryotic initiation

Answer 22

1) Ribosome recognizes shine-dalgarno sequence. 3’ end of the ribosome has a pyrimidine rich sequence that recognizes purine rich segment on 5’ end of mRNA.

Question 23

30S initiation complex components

Answer 23

1) 3x IF-x 2) GTP 3) fmet-tRNAfmet, mRNA, and 30S ribosomal subunit.

Question 24

EFTu

Answer 24

elongation factor that brings tRNA to ribosome; formal group inhibits fmet-tRNAmet from interacting with EFTu; formal groups only in prokaryotes.

Question 25

What two regions distinguish initiator met tRNA from other tRNAs?

Answer 25

3x GC region; C_A (non-bP) region.

Question 26

IF-3

Answer 26

initiation factor 3; binds to E site of the ribosome; prevents aminoacyl-tRNA from binding to E site and prevents 50s from binding to the 30s

Question 27

IF-1

Answer 27

binds 30S subunit in A site. prevents tRNA from binding to the A site

Question 28

Pre-initiation complex formation Prokaryotes

Answer 28

1) IF-3 binds the E site of the 30S subunit, IF-1 binds the A site, mRNA is recruited with the help of the 16s RNA (recognizes the shine delgarno sequence) 2) IF-2-GTP binds 30S and recruits fmet-tRNAmet

Question 29

70 Initiation complex

Answer 29

1) As soon as the initiator tRNA arrives in the P site, IF-3 leaves, which allows the 50S to bind to the 30S 2) 50S acts as a GAP hydrolyzing IF-2-GTP causing IF-2 and IF-1 to leave.

Question 30

Eukaryotic 40S initiation complex/transcription initiation overview.

Answer 30

1) EIF-1 binds E site; EIF1A binds to A site; EIF3 binds to the backside of the 40S subunit 2) EIF4F preps mRNA; EIF-2 binds to tRNA and guides it to P site along with EIFB-GTP and EIF-1A (In this case, the initiator tRNA is not guided by the mRNA transcript. 3) EIF4F complex aids in the loading with the entire ribosomal complex. 4)codon-anticodon interaction causes GTP hydrolysis of EIF2`

Question 31

EIF4F complex`

Answer 31

Complex that binds to mRNA before loading onto mRNA. has a segment that binds to the 5’ cap (EIF4E), the polyA tail (PABP) another just makes contact, and one has ATPase activity. ATPase activity helps complex weave through mRNA until it finds start codon AUG for anticodon-codon interation.

Question 32

PolyA binding protein

Answer 32

(PAPB) Binds polyA tail and EIF4 to stabilize the ribosome mRNA interaction for translation.

Question 33

Elongation Process in prokaryotes

Answer 33

1) EF-Tu binds aminoacyl-tRNA and GTP 2) complex binds to A site, GTP is hydrolyzed and EF-Tu GDP complex dissociates. 3) 23S subunit forms catalytic site. Nucleophillic attack of AA in A site to AA in P site 4) EF-G-GTP causes translocation of tRNA in A site to the P site.

Question 34

EF-G-GTP

Answer 34

Protein that binds to the tRNA in A site causes activation of GTPase ability, pushes tRNA in A site into the P site.

Question 35

Release factors

Answer 35

Bind to interaction with stop codons; causes hydrolysis of peptide chain.

Question 36

IRES proteins

Answer 36

binds to the 5’ cap of the mRNA transcript leading to the ribosome binding to the mRNA