9: Mechanics of Translation in Prokaryotes and Eukaryotes

tRNA

Transfer RNA that carries amino acids to ribosomes.

Aminoacyl t-RNA synthetases

Enzymes that attach amino acids to tRNA.

what is the Shine-Dalgarno sequence?

what is it complementary to?

Purine-rich region in prokaryotic mRNA for initiation.

is complementary to the initiator sites of mRNA

the start signal in prokaryotic mRNA is?

AUG or GUG

tRNAfmet

Formylated tRNA for initiating polypeptide chains.

recognises AUG and GUG

tRNAmmet

Non-formylated tRNA for internal methionine residues.

recognises AUG only

evidence of protein factors in initiation

• protein synthesis requires free 30S subunit

• if 30S subunits are in high salt = lose ability to initiate protein synthesis

• removing salt = activity restored

• chromatography of high salt supernatant = IF-1, IF-2 and IF-3

Initiation factors (IFs)

Proteins required for initiation of translation.

IF-1

Binds A site, directs fmet-tRNA to P site.

IF-2

Forms ternary complex with fmet-tRNA and GTP.

delivers this complex and mRNA to the partial P site in 30S subunit-mRNA complex

triggers GTP hydrolysis when 50S joins complex

the now complex does not recognise met-tRNA or any aa tRNA for elongation

IF-3 does what?

Prevents 30S subunit from binding 50S subunit prematurely - before correct mRNA and initiator tRNA are in place (in prokaryotes)

30S initiation complex

Complex formed by 30S subunit and initiation factors.

70S initiation complex

Complex formed when 50S joins 30S initiation complex.

GTP hydrolysis

Energy-releasing reaction during translation initiation.

elongation happens in what 3 steps?

1. codon-directed binding incoming aminoacyl-tRNA

2. peptide bond formation

3. translocation of ribosome along mRNA (5’ to 3’ direction)

Codon-directed binding

Process of matching tRNA to mRNA codons.

Peptide bond formation

Linking amino acids via peptide bonds during elongation.

Translocation

Movement of ribosome along mRNA during elongation.

Peptidyl transferase

Ribozyme that catalyzes peptide bond formation.

peptide bond formation by peptidyl transferase

1. fMet-tRNA is initially in the P site

2. new aminoacyl-tRNA enters A site

3. amino acid group attack fMet in the P site

4. fMet is transferred to A site

peptide bond is formed from moving peptide chain from P site to A site.

EF-Tu

is heat ______

Elongation factor that binds incoming aminoacyl-tRNA.

unstable

Diphtheria toxin inhibits ______ synthesis by acting during _________

protein

elongation

Overall Keq ~1

Indicates no net energy input required for translation.

Eukaryotic mRNA

Has distinct initiation and termination regions compared to prokaryotic.

Prokaryotic mRNA

Contains specific sequences for translation initiation.

EF-Ts

it is heat ____

Facilitates GDP to GTP exchange for EF-Tu.

stable

Cryo-EM

Technique for visualizing ribosome-tRNA complexes.

A/T site

Site for aminoacyl-tRNA during translation.

P site

Peptidyl site where tRNA holds growing peptide.

E site

Exit site for deacylated tRNA.

GTP hydrolysis

Process that triggers EF-Tu-GDP release.

Peptide bond formation occurs when?

Occurs after EF-Tu-GDP dissociates.

Translocation

Ribosome movement along mRNA by one codon.

EF-G/GTP acts in bacteria & does?

what does the same in eukaryotes?

Elongation factor that facilitates ribosome translocation.

EF-2

N-terminal region of EF-G mimics what and why?

Mimics tRNA structure for effective binding.

Sarcin/ricin loop

Part of 23S rRNA involved in EF-G binding.

mechanism of translocation

1. EF-G/GTP binds to pre-translocation ribosome

2. tRNA-like domain interacts with 30S close to partial A site

3. GTP hydrolysis = conformational change in EF-G = forces arm deep in 30S

4. peptidyl tRNA forced from A site to P site carrying mRNA and deacylated RNA

5. ribosome move along mRNA by length of one codon

Probability of error-free protein synthesis

p = (1-ɛ)n, where ɛ is frequency of inserting a wrong amino acid.

Elongation process

Involves EF-Tu-GTP and aminoacyl-tRNA delivery.

fMet-tRNAf

First tRNA in prokaryotic protein synthesis.

Initiation factors

Proteins IF-1, IF-2, IF-3 aid in translation start.

Labile ester bond

Bond between tRNA and amino acid, protected by EF-Tu.

Decoding centre

Region where codon-anticodon matching occurs.

Pre-translocation ribosome

Ribosome state before tRNA movement.

GTPase-associated centre (GAC)

Region involved in GTP hydrolysis during translation.

Translocation

Process requiring elongation factor EF-G/GTP.

Termination is simply…

Transfer of polypeptide to water ends translation.

termination steps [4]

1. release factors bind to vacant A site

2. peptidyl transferred to water rather than aminoacyl tRNA

3. hydrolysis of RF3-GTP to GDP dissociates everything

Initiation Factors

Proteins aiding the start of translation.

IF-1

Prevents premature tRNA binding to A site.

IF-2

Guides fMet-tRNAfMet to 30S subunit.

IF-3

Prevents premature 50S subunit association.

eIF1

Guides Met-tRNAi to 40S subunit.

eIF2B

Facilitates binding of initiation factors.

eIF3

First binder in eukaryotic translation initiation.

eIF4A

RNA helicase unwinding secondary structures.

eIF4B

Binds mRNA, facilitating scanning process.

eIF4E

Binds 5' cap of mRNA.

eIF4G

Links eIF4E and pol(A) binding protein.

eIF5

Promotes dissociation of initiation factors.

eIF6

Promotes dissociation of 80S into subunits.

AUG Codon

Common initiation codon for protein synthesis.

tRNAimet

Special initiator tRNA for eukaryotic translation.

Kozak Sequence

Sequence surrounding AUG for translation initiation.

Cap Binding Complex

eIF-4F complex binding to mRNA cap.

PAB1

Interacts with eIF4G and eIF4E at cap.

Internal Ribosome Entry Sites (IRES)

Alternative translation initiation lacking 5' cap.

Picornaviruses

Viruses using cap and IRES-dependent initiation.

IRES

Internal Ribosome Entry Site; allows translation independent of cell cycle.

Eukaryotic release factor

Mimics tRNA's acceptor stem structure for polypeptide release.

Peptidyl transferase center

Ribosomal site where peptide bond formation occurs.

Kozak sequence

Sequence that facilitates recognition of start codon.

Met-tRNA

Methionine-carrying tRNA pre-bound to ribosomal subunit.

eIF4E

Initiation factor that binds to the 5' cap.

Polysomes

Multiple ribosomes translating a single mRNA simultaneously.

Hydrolysis of ester bond

Water molecule cleaves bond, releasing polypeptide.

Translational control mechanisms

Regulation of translation via initiation and elongation factors.

Autogenous regulation

Gene product binds mRNA to prevent its translation.

Differential stability of mRNA

Variability in mRNA lifespan affects protein synthesis.

eIF2a phosphorylation

Regulates initiation of translation in eukaryotes.

eIF4E phosphorylation

Target for cancer drugs, regulates translation initiation.

R-protein synthesis

Regulated by growth rate and coupled to rRNA synthesis.

Operons

Groups of genes transcribed together, often in prokaryotes.

Iron-response element (IRE)

Sequence that regulates mRNA translation based on iron levels.

Aconitase

Protein that binds IRE under iron starvation conditions.

Ferritin

Cytosolic protein that stores iron, preventing toxicity.

Transferrin receptor

Cell surface protein for iron uptake regulation.

mRNA decay

Process of mRNA degradation after poly(A) tail shortening.

Decapping

Removal of the 5' cap leading to mRNA degradation.

Endonuclease cleavage site

Location in 3' UTR where mRNA is cleaved.

EF-Tu-GTP

Elongation factor that brings aminoacyl tRNA to ribosome.

EF-G/GTP

Elongation factor required for ribosome translocation.

Termination of translation

Process where polypeptide is transferred to water.