Protein Metabolism I Flashcards Preview

Biochemistry 431 > Protein Metabolism I > Flashcards

Flashcards in Protein Metabolism I Deck (36):
1

aminoacyl-tRNA synthetases

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

2

Stop Codons

UAA, UAG, UGA

3

Wobble Hypothesis

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

4

Why is the wobble effect hypothesis important?

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

5

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

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

6

Aminoacyl tRNA synthetase Reaction

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

7

Class I aminoacyl-tRNA synthetases

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

8

Class II aminoacyl-tRNA synthetases

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

9

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

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

10

Aminoacyl-tRNA synthetase sites

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)

11

Specificity of aminoacyl-tRNA synthetases to tRNA

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

12

Where does high fidelity of tRNA synthetases come from?

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

13

Editing pre-transfer

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

14

Editing post-transfer

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

15

Dbp5

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

16

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

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

17

Initiation of transcription in Eukaryotes

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

18

Pre-rRNA transcript

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.

19

Puromycin

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

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GAPs

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

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GEF

guanine-nucleotide exchange factors; recharge GDP to GTP

22

Prokaryotic initiation

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.

23

30S initiation complex components

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

24

EFTu

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

25

What two regions distinguish initiator met tRNA from other tRNAs?

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

26

IF-3

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

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IF-1

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

28

Pre-initiation complex formation Prokaryotes

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

29

70 Initiation complex

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.

30

Eukaryotic 40S initiation complex/transcription initiation overview.

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`

31

EIF4F complex`

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.

32

PolyA binding protein

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

33

Elongation Process in prokaryotes

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.

34

EF-G-GTP

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

35

Release factors

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

36

IRES proteins

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

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