Translation in Prokaryotes Flashcards

Flashcards for reviewing Translation in Prokaryotes and Eukaryotes

Translation

The process by which the mRNA sequence is decoded into a polypeptide (protein).

Ribosomes

Machinery for protein synthesis, composed of 30S and 50S subunits.

30S (small) subunit

Decodes mRNA during translation

50S (large) subunit

Catalyzes peptide bond formation during translation.

tRNA molecules

Carry specific amino acids; match codons via anticodon loops.

Initiator tRNA (fMet-tRNAᶠᴹᵉᵗ)

Starts the translation with N-formylmethionine.

GTP

Required for initiation, elongation, and termination steps.

ATP

Used during tRNA charging by aminoacyl-tRNA synthetases.

fmet-tRNAi

Prokaryotic Initiator tRNA

Shine-Dalgarno Sequence (SD Sequence)

A purine-rich sequence (typically: AGGAGG) located upstream (~6–10 nucleotides) of the start codon (AUG) on prokaryotic mRNA.

5' UTR

Found in the 5' untranslated region (5' UTR) of the mRNA.

16S rRNA

Part of the 30S ribosomal subunit that is complementary to Shine-Dalgarno Sequence

A Site – Aminoacyl Site

Entry point for new aminoacyl-tRNA (charged tRNA).

P Site – Peptidyl Site

Holds the tRNA with the growing polypeptide chain.

E Site – Exit Site

Holds the now-empty tRNA after its amino acid has been added to the chain.

IF1

Prevents tRNA binding to A site/Assists IF3.

IF2

Binds fmet-tRNAi and GTP.

IF3

Aids binding of mRNA; releases 30 S subunit from inactive ribosome.

EF-Tu

Binds aminoacyl-tRNA & GTP.

EF-Ts

Displaces GDP from EF-Tu.

EF-G

Translocation of ribosome.

RF1

Recognizes UAA and UAG stop codons.

RF2

Recognizes UAA and UGA stop codons.

RF3

Binds GTP and catalyzes release of RF1 and RF2.

Elongation: First Step

Aminoacyl tRNA binds to a complex of elongation factor Tu that also carries GTP.

Peptide Bond Formation

Catalyzed by peptidyl transferase, a ribozyme activity of 23S rRNA (not a protein enzyme).

Translocation in Bacteria

Ribosome moves one codon toward the 3’-end of the mRNA.

Conformational Mimicry: EF-G vs. EF-Tu/tRNA

Structurally mimics the EF-Tu–tRNA complex.

Termination is Signaled by a Stop Codon

Hydrolyze terminal peptide- tRNA bond, Release peptide and tRNA from ribosome, Cause subunits of ribosome to dissociate so that initiation can begin again.

Ribosome recycling factor (RRF)

Cooperates with EF-G and IF3 to dissociate the large and small subunits.

Coupling of Transcription and Translation in Bacteria

Translation begins before transcription ends.

Streptomycin

Causes misreading of the mRNA. Mutations in protein S12 or at C912 in 16S rRNA confers resistance.

Tetracycline

Inhibits binding of aminoacyl- tRNA to the 30S subunit.

Chloramphenicol

Inhibits peptidyl transferase.

Initiation in Eukaryotes

eIFs help mRNA recognition via the cap structure; 40S ribosome binds and scans for AUG; 60S ribosome joins to form the functional ribosome.

Elongation in Eukaryotes

eEFs (eEF1A, eEF1B, eEF2) assist in tRNA delivery, peptide bond formation, and ribosome translocation.

Termination in Eukaryotes

eRF recognizes all stop codons; ABCE1 helps ribosome dissociation and release of the polypeptide.

elF1

Binds to the E site of the 40S subunit; facilitates interaction between elF2-tRNA-GTP ternary complex and the 40S subunit

elF1A

Homolog of bacterial IF-1; prevents premature binding of tRNAs to A site

elF2

GTPase; facilitates binding of initiating Met-tRNA Met to 40S ribosomal subunit

elF2B*, eIF3

First factors to bind 40S subunit; facilitate subsequent steps

elF4F

Complex consisting of eIF4E, eIF4A, and eIF4G

eIF4A

RNA helicase activity; removes secondary structure in the mRNA to permit binding to 40S subunit; part of the eIF4F complex

eIF4B

Binds to mRNA; facilitates scanning of mRNA to locate the first AUG

eIF4E

Binds to the 5' cap of mRNA; part of the eIF4F complex

eIF4G

Binds to eIF4E and to poly(A) binding protein (PABP); part of the eIF4F complex

elF5*

Promotes dissociation of several other initiation factors from 40S subunit as a prelude to association of 60S subunit to form 80S initiation complex

elF5b

GTPase homologous to bacterial IF-2; promotes dissociation of initiation factors prior to final ribosome assembly

eIF-3 & eIF-1A

Blocks premature binding of subunits.

eIF-6 60 S

Blocks premature binding of subunits.

eIF-2 + GTP

met-tRNAi 40 S

Regulation of eIF-2

eIF-2 (α, β, γ subunits).

eIF-2α

Phosphorylated by at least 4 kinases (HCR, PKR, PERK, and GCN2).

Phosphorylated eIF-2α

Inhibits eIF-2B that aids in the GTP-GDP exchange of eIF-2.

GAP

GTPase Accelerating Protein

GDI

GDP Dissociation Inhibitor

eIF-5

Acts as a GAP for eIF-2 in the 40S initiation complex.

eIF-5

Acts as a GDI for eIF-2 as the 40S and 60S subunits come together.

eEF-1α + GTP

(EF-Tu)

eEF-1βγ

(EF-Ts)

eEF-2 + GTP

(EF-G)

eRF1

Recognizes all three stop codons: 1. UAA 2. UAG 3. UGA

Cycloheximide

Peptidyltransferase.

Puromycin

Chain termination.

Protein Translational Repressors

Function: In prokaryotes, translational repressors regulate gene expression by preventing the translation of mRNA in operons, thus controlling the synthesis of proteins.

Small Noncoding RNAs in Bacteria

NcRNAs can base-pair to mRNAs and change their translation efficiency and/or their stability, or they can bind to proteins and modulate their activity.

Signal Peptide/SRP Mechanism

Temporary Inhibition of Translation (Secreted Proteins)

Riboswitches

Direct (protein-free) binding of metabolite to RNA; Metabolite-dependent regulation of genes.

Hydrophobic effect

Nonpolar groups and water must be segregated.

Molecular Chaperones

Binds nascent polypeptide chains and aids in folding (required of most proteins).

Molecular Chaperones

Up regulated during heat shock and initially called heat shock proteins

Hsp60 (chaperonin-60) family

Gro EL (bacterial cytosol).

Hsp70 (stress-70) family

Hsp 70 (mammalian cytosol).

Hsp90 (sress-90) family

Hsp83 (eukaryotic cytosol).

Phosphorylation

Targeting Serine, threonine by Protein Kinase A

Acetylation

Example of targets: Lysine on Histones, tubulins, p53

Methylation

Example of targets: Lysine, arginine on Histones

Ubiquitin Pathway

Complex is a substrate for a protease complex: the 26S proteasome.

E1

Ubiquitin activating enzyme

E2

Ubiquitin conjugating enzyme

E3

Ubiquitin protein ligase

Proteasome Cap

Recognizes polyubiquitinated proteins targeted for degradation; Removes ubiquitin chains for recycling; Unfolds proteins using ATP-dependent ATPases; Opens the gate of the 20S core for substrate entry; Translocates the unfolded protein into the 20S core for degradation; Ensures specificity and quality control in protein degradation.