BMED 5207 Unit 3 slides

What does translation of mRNA require?

3 types of RNA(mRNA, tRNA, rRNA), Energy, proteins

What types of RNAs are required for mRNA?

mRNA, tRNA, rRNA

What energy sources are used in translation of mRNA?

ATP, GTP

What proteins are needed for translation of mRNAs?

Ribosomal proteins, IF, EF, RF

What are IF proteins?

Translation initiation factors

What are EF proteins?

translation elongation factors

What are RF proteins?

release factors

What are the three phases of translation?

initiation, elongation, termination

What does mRNA do?

carries the genetic information from the nucleus to the site of protein synthesis in the cytosol

What does tRNA do?

carries the amino acids to the site of protein synthesis and deciphers the codes

What are rRNA?

structural components of ribosomes

How many correct reading frames do each mRNA molecules have in eukaryotes?

one

What is the reading frame defined by?

the first strong AUG

What is the sequence that makes the AUG “strong”?

[A/G]NNAUGG

What decipher the codons of mRNA?

tRNAs

Where are tRNAs synthesized and modified?

in the nucleus

What are tRNA modifications?

base modifications, shortening of 5’ end and adding CCA to the 3’ end

What happens when tRNAs are exported to the cytoplasm?

they are aminoacylated

What are the loops of tRNAs important for?

interactions with proteins and the translated mRNA

How specific is the attachment of the amino acids to their respective tRNAs?

highly specific, each amino acid has its own aa-tRNA synthetase that recognizes the aa and all corresponding tRNAs

Proofreading activity of aminoacyl-tRNA synthetases

ensures correct charging of tRNAs, it cleaves the bond between the aa and the incorrectly charged tRNA

Wobbling position of codons

first and second bases of the codon form standard base pairs, the 3rd base of th codon can form non-standard pairs with the anticodon

Which two amino acids don’t have more than one codons that encode them?

Met and Trp

Why is the genetic code degenerate?

2 or more codons encode a single amino acid (except for met and trp)

how does the translation machinery select Met-tRNA vs Ile-tRNA?

the 3’-UAU-5’ anticodon is modified soon after transciption to 3’-UAΨ-5’ becasue Ψ cannot form base pair with G in the codon, only with A

What rRNA has catalytic activity?

28S

What is the catalytic activity of the 28S rRNA critical for?

peptide bond formation during translation

Subunits of ribosomes

60S and 40S

Where are rRNAs produced?

the nucleolus

Where does the ribosome assemly start?

the nucleolus

Translation initaiton I

formation of the 43S preinitiation complexs; steps 1-4

first step of translation initiation

eIF2B exchanges GDP to GTP on eIF2

second step of translation initiation

eIF2-GTP binds Met-tRNAi; a ternary complex is formed (the 3 components are eIF2, GTP, and Met-tRNA)

third step of translation initiation

the ternary complex is recruited to the P site of the 40S subunit of the ribosome, which bins to some other eIFs too

fourth step of translation initiaiton

eIF5 also joins to comples, which completes formation of 43S preinitiation complex

What is a “scanner” for AUG in the mRNA

43S

Translation initiation II

mRNA activation; steps 5-8

fifth step of translation initiation

PABPC is recruited to the polyA tail

sixth step of translation initiation

eIF4 complex assembles at the 5’ cap

seventh step of translation initiation

the eIF4 complex binds PABPC

eighth step of translation initiation

the eIFA subunit of the eIF4 complex is a helicase that unwinds the secondary structures in the 5’ UTR using ATP

What makes the recruitment of the 43S compolex possible at the cap?

unwinding the secondary structures in the 5’ UTR

Translation initiation III

assembly of the 80S ribosome; steps 9-11

ninth step of step of translation initiation

the 43S preinitiation complex is recruited to the cap and it scans for the first strong AUG

tenth step of step of translation initiation

AUG recognition causes GTP hydrolysis by eIF2, the 48S initiation complex is formed, this is check point, process becomes irreversible under physiological conditions

eleventh step of step of translation initiation

60S binds to the smaller ribosomal unit, correct binding between the large and small ribosomal subunits triggers the hydrolysis GTP

Result of translation initiation

a correctly positioned 80S complex with Met-tRNA at th eP site and 80S-mRNA-tRNAiMet complex is ready for elongation

How fast is the polypeptide chain elongation?

2-5 aa per sec

first step of polypeptide chain elongation

eEF1-alpha binds GTF and an aa-tRNA, a terniary complex is formed for elongation

second step of polypeptide chain elongation

the codon at the A site recruits the complementary aa-tRNA-eEF1alpha

third step of polypeptide chain elongation

correct codon-anticodon pairing induces hydrolysis of GTP by eEF1alpha, leading to conformation change in the ribosome and eEF1alpha is released

fourth step of polypeptide chain elongation

in the new conformation state of th eribosome, the two aa-tRNAs are close to each other and the large rRNA catelyzes the formation of the peptide bond

fifth step of polypeptide chain elongation

the 80S complex binds eEF2-GTP, the GTF is hydrolyzed by EF2 and this provides the energy for translocation where the ribosome moves forward by 3 bases and rRNAi ends up in the E sites, now the dipeptidyl-tRNA is at the P site, and the ribosome is ready to bind the next ternary complex at the A site

structure of eRF1 protein

similar to that of a tRNA but is not an RNA

first step of termination of translation

eRF1 binds to eRF3-GTP, another ternary complex is formed

second step of termination of translation

eRF1-eRF3-GTP recognizes the stop codon

third step of termination of translation

GTP is hydrolyzed by eRF3

fourth step of termination of translation

GTP hydrolysis by eRF3 causes conformation changes and this leads to the cleavage of the peptidyl-tRNA bond and the release of the polypeptide

fifth step of termination of translation

a few eIFs and the ATPase ABCE1 are required for the dissociation of ribosomal subunits from the mRNA, for the dissociation of the tRNA from the P site and for the release of eRF1 and eRF3

How many times can most mRNA molecues be translated?

several times, many ribosomes can translate a single mRNA molecule nearly simultaneously

What is the poly(ribo)some?

mRNA with many ribosomestructues

What is the mRNA structure/codon that recruits the 43S complex?

5’ cap

60S is recruited to the mRNA to which mRNA structure/codon?

start codon

The small and large ribosomal subunits (40s and 60S) dissociate from the mRNA at which mRNA structure/codon?

stop codon

Why can’t the start codon be very close (<8 bases) to the 5’ cap of the mRNA?

When 43S is recruited to the 5’ cap, the P site is positioned beyond the first 8 bases,so any AUG within the first 8 base region will be missed by the 43S complex

Which molecules/protiens/complexes are components of the ternary complex of translation initiation?

eIF2, GTP, Met-tRNAi

What would potentially happen to the 80S ribosomal complex when it “arrives” to the end of a truncated mRNA with no stop codon?

the 40S and 60S ribosomal subunits do not dissociate from each other and the mRNA and the produced polypeptide chain is not released from tRNA; the tRNA binds to the last compolete codon of the mRNA in the P site; dissociation would require recognition of the stop codon by the eRF1-eRF3-GTP complex and 5’ to 3’ movement o fthe ribosome requires recruitment of an aa-tRNA to the A site

What does the regulation of translation provides the cell with?

the plasticity that is needed to respond rapidly to changes in the environment

Regulation of translation is especially important during what?

cellular stresses (ER stress, not enough amino acids and other metabolic stresses, viral infections, apoptosis)

What are the levels of regulation of mRNA translation?

global regulation and mRNA-specific regulation through interactions between proteins and unique structures in mRNAs

Global regulation

phosphorylation of eIF2, phosphorylation of eIF4E bindng protein (4E-BP), proteolytic cleavage of eIF4G and PABPC

mRNA-specific regulation through interactions between proteins and unique structures in mRNA

upstream open reading frame (uORF) and internal Ribosome Entry Site (IRES)

eIF2 phosphorylation by eIF2 kinases

phosphorylation of alpha subunit of the heterotrimeric protein eiF2 at a serine residue blocks the GDP to GTP exhange reaction by eIF2B, which is a GEF of eIF2; the end result is that the concentration of the ternary complex of translation initiation is reduced

WHat are the eIF2 kinases activated by?

heme depletion, dsRNA, ER stress, amino acid depletion

What eIF2 kinase is activated by ER stress?

PERK

What eIF2 kinase is activated by amino acid depletion?

GCN2

When is PERK activated?

when unfolded proteins sequester the chaperone BiP in the ER lumen

first step of PERK activaiton

ER stress/ accumulation of misfolded proteins in ER lumen

second step of PERK activation

the ER chaperone BiP is sequestered by misfolded proteins in the lumen of the ER

third step of PERK activation

PERK releases BiP; the release of BiP by Perk leads to PERK dimerization and activation of its kinase domain; PERK phosphorylates itself an deIF2

fourth step of PERK activation

Phosphorylation of eIF2 causes global inhibition of translation but translation of the ATF4 mRNA increases

fifth step of PERK activation

ATF4 is a transcription factor; ATF4 induces expression of many other stress response proteins

How is GCN2 activated?

by non-acylated tRNAs

What increases th econcentration of non-acetylated tRNAs in the cytosol?

animo acid starvation

What activates the kinase domain of GCN2?

Histidyl-tRNA sythetase-related domain binding with non-acetylated tRNAs

Process of inhibiting protein synthesis and expression of stress response genes with eIF2 kinases

eIF2 gets phosphorylated with eIF2 kinases to eiF2-P; eIF2-P inhibits general protein synthesis and increases translation of ATF4 mRNA; increase translation of ATF4 leads to the induction of expression of many stress response genes

GADD34-dependent negative feed-back regulation of translational repression

ATF4 translation upregulates GADD34 transcription and the GADD34 protein increases; GADD34 provides a scaffold for interaction of PP1 and P-eIF2alpha and dephosphorylates P-eIF2alpha

How can ATF4 mRNA be translated when eIF2 is phosphorylated

the ATF4 mRNA contains 2 uORFs besides the ATF4- coding sequence (ORF); first uORF is very short, second uORF overlaps the ORF of ATF4, second uORF and ORF of ATF4 are not in frame

Model for uORF-dependent regulation of ATF4 translation

40S remains on th emRNA at the stop of uORF1 and it continues to move along the mRNA; 40S becomes competent for translation reinitiation faster if the concentraiton of the ternary complex (TC) is high in the cytosol

No stress with uORF-dependent regulation of ATF4 translation

high TC concentration, 40S becomes competent for reinitiation upstream of uORF2 by binding the TC

Stress with uORF-dependent regulation of ATF4 translation

low TC concentration, 40S becomes competent for reinitiation downstream of uORF2 and reinitiate at the AUG of the 3rd ORF of ATF4

How can 40S remain on the mRNA at the stop of uORF1?

at the start codon, when 60S binds to 48S, it takes a few seconds for some of the eIFs to fall off from the assembled ribosome and if the ribosome arrives to the stop codon before eIFs fall off, 40S will not dissociate from the mRNA at stop, and the 40S continues to move towards the 3’ end of the mRNA

Stimulation of translation through 4E-BP phosphorylation

the non-phosphorylated 4E-BP inhibits/represses translation; when nutrients are available, mTOR phosphorylates 4E-BP and 4E-BP is released, allowig translation initiation

What does mTOR stand for?

metazoan target of rapamycin

What is mTOR?

a kinase

What is an inhibitor of mTOR?

rapamycin

What promotes mTOR?

growth hormones, amino acids, O2

What does amino acid deprivation regulate?

availability of ternary complex through GCN2 and eIF4G through mTOR

What does IRES stand for?

internal ribosome entry site

What is IRES?

an mRNA sequence with strong 3D structure that recruits the 43S complex directly , without using the 5’ cap or eIF4E