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Aims

• Identification of the presence of a STOP codon in the A site

• Release of the completed polypeptide chain from the tRNA and ribosome

• Recycling of the mRNA and the ribosome

Translation Termination: Overview

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Translation Termination in Prokaryotes

The mechanism is virtually identical in Eukaryotes

Release factors RF1 and RF2 recognize all three STOP codons via binding to the

A-site: • RF1 → UAG

• RF2 → UGA

• RF1-RF2 complex → UAA

A conserved GGQ tripeptide motif in A site bound RF1 and RF2 becomes

positioned adjacent to the 3‘-end of the polypeptidy-tRNA and triggers hydrolysis of

the ester bond between polyeptide and tRNA

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Translation Termination in Prokaryotes Release factor RF3-GDP binds to RF1/RF2/RF1-RF2 located in the A-site. This

interaction stimulates RF3 to exchange GDP aganst GTP.

The factor binding center stimulates the GTPase activity of RF3, which

dissociates from the ribosome as RF3-GDP complex.

Release of the polypeptide chain triggers conformational changes in the ribosome,

which leads to dissociation of RF1/RF2/RF1-RF2, while RF3-GTP binds to the

factor binding center in the large ribosomal subunit.

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Ribosome Recycling in Prokaryotes To initiate disassembly of the remaining ribosome-tRNA-mRNA complex, the

ribosome release factor (RRF) binds to the empty A site (note: RRF is a protein

mimic of a tRNA molecule)

A site-bound RRF recruits EF-G-GTP to the ribosome. As during elongation, GTP

hydrolysis triggers translocation of the ribosome. This leads to movement of RRF

to the P site and the release of the tRNAs from the E and P sites.

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Ribosome Recycling in Prokaryotes A site-bound RRF recruits EF-G-GTP to the ribosome. As during elongation, GTP

hydrolysis triggers translocation of the ribosome. This leads to movement of RRF

to the P site and the release of the tRNAs from the E and P sites.

EF-G-GDP has low affinity to the A site ands RRF has low affinity to the P site and

thus both proteins dissociate from the ribosome.

IF3 binds to the E site, which triggers disassembly of the large and small ribosomal

subunits and the release of the mRNA.

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Enhancing Re-initiation of Translation by

Circularization of eukaryotic mRNA Inititation factor eIF4G has strong affinity for the polyA-tail of mRNA through

interaction with the polyA binding proteins → circularization of mRNA

The proximity of the 3‘- and 5‘-ends of mRNA foster the immediate re-use of the

small ribosomal subunit that was released during termination of translation.

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Prokaryotic and eukaryotic Translation Factors

Prokaryotes Eukaryotes

Initiation IF1 eIF1A

IF2 eIF2

IF3 eIF1, eIF3, eIF5

--- eIF4A, eIF4B, eIF4E, eIF4G

Elongation EF-Tu (GDP/GTP bexhange

factor EF-Ts)

eEF1

EF-G eEF2

Termination RF1, RF2 eRF1

RF3 eRF3

RRF ABCE1 (ATPase activity)1

1T.E. Dever, R. Green (2012) The Elongation, Termination, and Recycling Phases of Translation in

Eukaryotes. Cold Spring Harb. Perspect. Biol. 4, a013706

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Antibiotics as Inhibitors of prokaryotic Translation

~40% of known antibiotics are inhibitors for different steps in translation by

specifically binding a component of the prokaryotic translation machinery.

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SsrA rescues Ribosomes from impaired mRNAs Incompletely translated or chemically damaged mRNAs may lack an in frame

STOP codon → ribosomes stall at these mRNAs and cannot become released by

the termination machinery.

Ala-charged SsrA RNA (a tRNA-mRNA hybrid molecule = tmRNA) in complex with

EF-Tu-GTP binds to the A site of stalled ribosomes

→ Elongation can continue normally using the SsrA sequence as codons.

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SsrA rescues Ribosomes from impaired mRNAs

An in-frame STOP codon on SsrA trigers termination → disassembly of the

ribosome and releasae of a tagged polypeptide that becomes rapidly degraded.

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Nonsense-mediated mRNA Decay After splicing, exon junction complexes (EJC proteins) become positioned 20-24 nt

upstream of each splice junction. Incorrectly spliced mRNAs may contain a

premature stop codon (see p. 106).

When a ribosome encounters a premature STOP codon, ECJ proteins are still

present on the mRNA, which leads to a recruitment of Upf proteins to the

ribosome. Ribosome-bound Upf activates the 5‘-decapping enzyme, which

initiates complete degradation of the mRNA by a 5‘→3‘ endonuclease.

Normal translation Nonsense-mediated mRNA decay

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Nonstop-mediated Decay When a STOP codon is missing in eukaryotic mRNA, the ribosome translates also

the polA-tail, which results in the attachment of multiple lysine residues at the

C-terminus of the polypeptide (AAA encodes Lys) and stalling of the ribosome at

the 3‘-end of the mRNA.

The stalled ribosome is bound by Ski7 (a eRF3 related protein), which recruits a

3‘→5‘ exonuclease complex (exosome) to the ribosome.

Ski7 triggers ribosome disassembly and release of the polypeptide chain. The

mRNA is degraded by the exosome and the poly-Lys-tagged protein by cellular

proteases .

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How long does E. coli need for

Biosynthesis of a 30 kDa protein?

*From: R. Milo, R. Philips, N. Orme “Cell Biology by the Numbers“, Garland Science, 2016.

• 30 kDa protein ≈ 273 amino acids (average: 110 Da/amino acid) ≈ 819 nt (codons)

≈ 1000 nt (including 5‘- and 3‘ UTRs)

• Transcription: RNA Pol links NTPs at a speed of 40-80 nt/s*

→ ~20 s to produce a 1 kb mRNA

• Translation: The ribosome links amino acids at a speed of ~20 amino acids/s*

→ ~15 s to produce a 273 amino acid polypeptide

→ TOTAL: It takes ~35 s to produce a 30 kDa polypeptide from activation of gene

expression to release of the polypeptide chain from the ribosome.

Folding of the protein into its native 3D structure takes usually <60 s*.