Eukaryotic Translation For Class Mtech

Eukaryotic Translation

Translation initiation

Phases of translation 1. Initiation 2. Elongation 3. Termination

Translation initiation in eukaryotes 1. Absolute requirement for cap on 5’ end (majority of RNAs) 2. For 95% of eukaryotic mRNAs translation begins at 5’-proximal AUG 3. Small ribosome subunit can scan in one dimension on RNA 4. mRNA secondary structures located in the 5’-untranslated region inhibit translation initiation

Kozak consensus sequence

5’

A GCC G CCAUGG

Marilyn Kozak identified this consensus sequence around the AUG start codon. It enhances initiation frequency

3’

Characteristics of mammalian translation factors Name

Function

eIF1A

Promotes Met-tRNA binding, ribosomal dissociation

eIF2

Binds Met-tRNA and GTP

eIF2α

Site of phosphorylation on Ser-51

eIF2B

Guanine nucleotide exchange factor for eIF2

eIF3

Dissociates ribosomes, promotes Met-tRNA and mRNA binding

eIF4A

ATPase, helicase, binds RNA

eIF4B

Binds RNA, promotes helicase activity

eIF4E

Cap-binding subunit, part of eIF4F complex

eIF4G

Binds eIF4A, eIF4E and eIF3 – acts as bridging factor

eIF5

Promotes GTPase with eIF2 and ejection of eIFs

eIF6

Binds to 60S ribosomes, promotes dissociation

Stages in translation initiation 1. eIF3 binds 40S ribosome - eIF6 binds 60S ribosome to keep them apart 2. eIF2 binds GTP + initiating methionine tRNA to form eIF2.GTP.mettRNA. This binds to 40S 3. RNA is loaded with factors at cap site; eIF4E (cap binding protein), eIF4B and eIF4A (unwind RNA) 4. 40S ribosome (+ eIF3 + ternary complex) binds to cap (interaction of eIF3 with eIF4G)

5. 40S ribosome scans along 5’-UTR (requires ATP) 6. When AUG is reached, eIF2.GTP is hydrolysed (possibly triggered by eIF5) which releases all factors from 40S subunit 7. 60S subunit can then bind to 40S subunit 8. eIF2.GDP must be recycled to eIF2.GTP by action of eIF2B

mRNA

Translation initiation in prokaryotes and eukaryotes Prokaryotic mRNA initiation occurs at AUG codons with properly spaced Shine-Delgarno sequence

5’

AUG

SD

AUG

initiation codon with Shine-Delgarno site

AUG

SD

internal Met codon does not have Shine-Delgarno site

AUG

3’

initiation codon with Shine-Delgarno site

Shine-Delgarno (SD) site consists of 3-9 contiguous bases in the mRNA that base pair with the 3’ end of 16S rRNA and is located optimally 5 nt upstream of the initiator codon

Eukaryotic mRNA initiation occurs ~90% of the time at the first AUG codon

5’ cap

AUG

AUG

first AUG codon downstream of the 5’ cap

internal Met codon

AUG

3’

Basic steps of translation initiation: joining of 40S and 60S AAAAAAAAA PABP

eIFs eIF4E

M

40S

GTP

UAC m7GpppG UACCAUGGAGCUUGGCCUGAUGCC A A U 28S rRNA, 5S rRNA, 5.8S rRNA, 49 proteins G 60S U A C

•joining of a large (60S) subunit to assemble a complete (80S) ribosome

Ternary complex formation eIF2

GDP

GDP-bound eIF2 cannot bind Met-tRNAiMet

guanine nucleotide exchange factor (GEF)

eIF2B eIF2

GDP

GTP

eIF2

GTP

Met

initiator methionyl tRNA

eIF2

GTP Met

ternary complex

GTPase activating protein (GAP)

eIF5 eIF3 eIF2 eIF1

GTP Met

multifactor complex (MFC)

43S complex formation eIF5 eIF3 eIF2 eIF1

GTP Met

eIF1A helps generate a pool of free 40S subunits, and eIF1A and eIF3 promote binding of the ternary complex to the 40S subunit

eIF5 eIF3 eIF2 eIF1

40S GTP Met

eIF1A

eIF4F binds the cap structure on the 5’ end of mRNAs

AAAAAAAAA PABP PABP eIF4G eIF4E

eIF4A m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

•eIF4 factors (along with eIF3) promote binding of the MFC/40S subunit to the mRNA •eIF4F = eIF4E (the cap-binding protein) eIF4A (the DEAD box, ATP-dependent RNA helicase that can unwind RNA secondary structure) eIF4G (an adapter molecule that binds both eIF4E and PABP as well as eIF3)

eIF4B stimulates the helicase activity of eIF4A

AAAAAAAAA PABP PABP eIF4G eIF4E

eIF4A eIF4B m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

•RNA secondary structure in the 5’ UTR, especially adjacent to the cap, impairs ribosome binding and/or scanning

Recruitment of the 43S complex to the 5’ end of the mRNA AAAAAAAAA PABP PABP

eIF4G eIF4E

eIF3

eIF5 eIF2 eIF1

40S GTP Met

eIF1A eIF4A eIF4B m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

•eIF3 interacts with eIF4G to recruit the 43S complex

Scanning of the 5’ UTR and AUG recognition

eIF4G

eIF3

eIF4E

eIF5 eIF2 eIF1

40S GTP Met

eIF1A eIF4A eIF4B m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

eIF4B

eIF4A

ATP

eIF4E eIF4G

ADP + Pi

eIF3

eIF5 eIF2 eIF1

40S GTP Met

eIF1A

m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

Conformational change, GTP hydrolysis, release of initiation factors, and assembly of the eIF5B GTPase

eIF3

eIF5 eIF2 eIF1

40S GTP Met

eIF1A

m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC eIF3 GTP hydrolysis by eIF2 requires eIF5 and possibly a conformational change triggered by the Met-tRNAiMet interaction with the 40S subunit

eIF2

GDP

eIF1

eIF5

40S Met

eIF5B eIF1A

m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC

GTP

Assembly of the 80S ribosome 40S Met

eIF5B eIF1A

m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC eIF1A

eIF5B

GTP hydrolysis by eIF5B serves as a final checkpoint for correct 80S assembly

40S Met

m7GpppGAUUCGAUACCAGGGAGCUUGGCACCAUGGC the 80S ribosome is now poised to elongate

60S

GDP

GTP

Summary of eukaryotic initiation factors name

subunits

function

eIF1

1

fidelity of AUG codon recognition, destabilizes aberrant initiation complexes

eIF1A

1

catalytically promotes Met-tRNAi binding to 40S; required for strong binding of 40S subunit to mRNA

eIF2

3

GTPase, escorts Met-tRNAi onto 40S subunit

eIF2B

5

guanine nucleotide exchange factor for eIF2

eIF3

11

scaffold for the cap binding complex, binds 40S subunit; stabilizing Met-tRNAi and preventing association with 60S subunit

eIF4A

1

RNA dependent ATPase; essential for binding of ribosomes to mRNA

eIF4B

1

RNA binding protein; promotes eIF4A activity

eIF4E

1

binds directly to the m7G cap

eIF4F

3

cap binding complex of eIFs 4A, 4E, and 4G

eIF4G

1

binds mRNA, PABP, eIF4E, eIF4A, and eIF3

eIF4H

1

similar to eIF4B

eIF5

1

AUG recognition and promote eIF2 GTPase activity

eIF5B

1

GTPase, mediates assembly of 80S from 40S and 60S

Messenger RNAs are translated on polyribosomes

Nutritional controls

2

Nutritional signals can control both the recognition of the mRNA and loading of the 40S subunit.

As the ribosome translocates, it moves in three nucleotide steps, ensuring that the frame defined by the AUG is used throughout translation

If the ribosome moves 1 or 2 (or 4 or 5) nucleotides this produces a frameshift

Translation Inhibitors are important antibiotics