MBII - L23 - Translation 2

8/8/2019 MBII - L23 - Translation 2

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Molecular Biology II Translation 2

Lecture [Page 1]

Ribosomes:

Prokaryotes Eukaryotes70S 80S

rRNA Proteins rRNA Proteins5S / 23S L1/2/3 (31 total) n Large Subunitp 5S / 5.8S / 28S L1/2/3 (50 total)16S S1/2/3 (21 total) n Small Subunitp 18S S1/2/3 (33 total)

Structure:

Both subunits are composed of long strands of RNA dotted with protein

Small subunit (E. coli) Large subunit (E. coli) ~1500nts Binds mRNA and ensures fidelity Flexible hatch opens allowing mRNA to enter mRNA then extends into decoding centre

~3000nts Binds tRNAs Contains the catalytic site Protein hold the RNA in shape RNA contains the catalytic activity Active site is at the centre of the complex

rRNAs form complex secondary structures including bulges hairpins etc.

23S rRNA has 6 domains

Peptidyl transferase centre (PTC) forms the peptide bond and is found in the centre of the 3D structure.

The ribosome is a ribozymeThe active site is made of RNA only nearest RNA 3 nearest protein 20

Ribozyme Mechanism:

Talk to ed about what needs to be learnt

Protein Synthesis:

Small subunit

Large subunit


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Lecture [Page 2]

1. InitiationThe initiation codon is AUG, which encodes methionine

All newly translated proteins have an N-terminal Met

The 1st Met is modified N-Formylmethionine

tRNAfMet is used for the 1st Met

Regular tRNAMet is used for the internal Met residues

Prokaryotes

Bacterial ribosomes have 3 sites that bind aminoacyl-tRNAs:

A (aminoacyl) site. Found on 30/50S. P (peptidyl) site. Found on 30/50S. E (exit) site. Found on 50S only.

Initiation in prokaryotes requires:

30S ribosomal subunit 50S ribosomal subunit mRNA transcript tRNAfMet IF-1/2/3 (Initiation Factors)

o IF-1 prevents premature binding of tRNAs to A siteo IF-2 forms a specific complex with tRNAfMet and GTP (also has GTPase activity)o IF-3 prevents 30S & 50S subunits from combining prematurely by binding to the 30So IF-3 complex is suitable for binding to mRNA via the RBS

GTP Mg2+

a) The 30S ribosomal subunit binds IF-1 & IF-3

b) mRNA binds to 30S subunit via Shine-Dalgarno sequence (aka [RBS] Ribosome Binding Site)

It base pairs with the 16S rRNA in the 30S subunit RBS is located before start codons and contains a consensus element (AGGAGG) that is complementary to

the 3 end of 16S rRNA

The initiation codon (AUG) is positioned at the P site, and is distinguished from other Met codons by itsproximity to the RBS in the mRNA.


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Lecture [Page 3]

c) tRNAfMet and IF-2-GTP are brought into the P site

This is the only site to which tRNAfMet can bind tRNAfMet is the only aminoacyl-tRNA that binds first to the P site The anticodon of the tRNAfMet pairs correctly with the mRNA initiation codon

d) The complex formed in c) combines with the 50S subunit

IF-2-GTP is simultaneously hydrolysed to GDP + Pi, causing a conformational change All 3 IFs then leave the ribosome

e) A functional ribosome, known as the initiation complex has been formed. It contains the mRNA and the initiating

tRNAfMet


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Lecture [Page 4]

Correct binding of the tRNAfMet to the P site is assured by 3 points of recognition:

Codon Anticodon interaction (AUG UAC) RBS (mRNA) 16S rRNA (ribosome) P site - tRNAfMet

Use of the RBS to initiate transcription allows bacterial mRNAs to encode several different proteins at one time.

Known as polycistronic mRNAs. e.g. Lac operon (LacZ/Y/A). Each ORF is preceded by a RBS, allowing ribosomes to

initiate internally on a polycistronic mRNA.

Eukaryotes

No ribosome binding site (RBS) More IFs (Initiation Factors): 9+ Ribosome positioning relies on the 5 cap and 3 polyA tail Ribosomes recognise 5 cap, then scan in the 5p 3 direction until AUG codon is reached, signalling the

start of a reading frame

The 3 polyA tail binds to a PAB(Poly A Binding protein)


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Lecture [Page 5]

The 5 cap binds to the eIF4E complexThe combined complex of mRNA, eIFs and PAB bind to the 40S subunit (above)

2. ElongationIf all the prerequisites in the initiation phase have been met, elongation can occur.

Prokaryotes

Elongation in prokaryotes requires:

Prokaryotic initiation complex Aminoacyl-tRNAs Elongation Factors(soluble cystolic proteins) GTP

a) Cells use 3 steps to add each amino acid to the growing chain.

I) The appropriate incoming aminoacyl-tRNA bind to

GTP-bound EF-Tu

II) The resulting complex binds to the A site of the 70S initiation

complex

III) GTP is hydrolysed and EF-Tu-GDP is released from the 70S

ribosome. EF-Tu-GTP complex is regenerated using EFTs

The steps are then repeated for the next amino acid

EF-Tu binds GTP, and all aa-tRNAs except for tRNAfMet/ tRNAMet

EF-Ts does not bind GTP, displaces GDP from EF-Tu

EF-Gbinds GTP, involved in translocation

b) A peptide bond forms between the two AAs that are bound by

their tRNAs to the A/P sites

23S rRNA acts as a peptidyl transferase (catalysespeptide bond formation)


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M

!

23S is a ribozym " I) Initiator fM " t is transferred from its tRNA to the amino group of the AA in the A site

P site contains an uncharged tRNA (lacking an amino acid) A site contains a dipeptidyl-tRNA The -NH2 of the AA in the A site acts as a nucleophile, attacking rhe carbonyl group of the peptide in the P

site. A peptide bond forms between the 2 AAs

II) The uncharged tRNA shifts so that its 3 & 5 ends are in the E site, and the dipeptidyl-tRNA

3 & 5 ends shift to the P site

Both anticodons remain in the A & P sites

c) The mRNA moves one codon length in the 5p 3 direction (translocation)

The dipeptidyl-tRNA anticodon and the 2ndmRNA codon associated with it move from the A to the P site The uncharged tRNA moves from the P to the E site, where the tRNA is released A third mRNA codon now lies in the A site


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Lecture [Page 7]

Movement of the ribosome along mRNA requires

EF-G (translocase) and the energy provided by

GTP hydrolysis

Eukaryotes

Has the same mechanism and similar EFs

eEF1/2/3Eukaryotic ribosomes do not have an E site,

uncharged tRNAs are expelled directly from the P

site

3. TerminationThe release of a completed peptide

No tRNA recognises UAA/UGA/UAG (stop codons)

Prokaryotes have release factors (RFs) that recognise these codons

RF1 UAA/UAG RF2 UAA/UGA RF3 Stimulates RF1/2

Eukaryotes have a single release factor (eRF) that recognises

stop codons

The release factors will encourage the ribosome to transfer the

peptide to an H2O rather than an aminoacyl-tRNA, causing the

protein to dissociate.

n


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Lecture [Page 8]

Inhibition of Translation:

Some toxins and antibiotics can inhibit translationPuromycin Similar to 3' end of tRNA, binds A site causing premature termination

Tetracyclines Block A site

Chloramphenicol Blocks peptidyltransferase

Cycloheximide Blocks eukaryotic peptidyltransferase

Streptomycin Causes misreading of codons

Translation Energetics:

EF-G and EF-Tu occupy the same binding site.

The distal domains 4 and 5 of each molecules have the same shape and size

For each amino acid

1 ATP is used to attach an amino acid to a tRNA 2 GTPs drive translation

o 1 for initiation with IF-2o 1 for elongation with EF-Tu

The translation process is highly conserved between prokaryotes and eukaryotes, the only major difference is in

initiation.

MBII - L23 - Translation 2

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