Lecture 7

Mechanism of Termination & Recycling Michael Altmann FS 2015

Institut für Biochemie und Molekulare Medizin

Polypeptide chain termination

Termination factors eRF1 eRF3

Termination mechanism

Ribosome recycling eRF3-PABP interaction; ribosome „channeling“ DOM34 & HBS1 ABCE1/RLI

80S ribosome structure at 3.0 Å

mRNA degradation

Termination & Recycling Factors

Protein name (prokaryotes)

Homolog (eukaryotes)

Properties

Class I release factors (RFs)

RF1/RF2

eRF1 49 kD

• RF1 recognizes UAA/UAG; RF2 recognizes UAA/UGA • Tripeptide motifs PXT in RF1 and SPF in RF2 confer specificity for the codons UAG or UGA • The tripeptide motif GGQ which is universally conserved in RFs triggers peptide-tRNA hydrolysis through the contact with the peptidyl-transferase center (PTC) on the ribosome • eRF1 is omnipotent (recognizes all three stop codons) • Q in GGQ motif becomes methylated when in eRF1/eRF3/GTP complex • In yeast, eRF1 is also known as Sup45p

Class II release factors (RFs)

RF3 eRF3 76.5 kD

• GTPase; facilitates removal of RF1/RF2 from post-termination complexes • RF3-GTP resembles EFTu-GTP-tRNA & EF-G • GTPase; helps eRF1 to ensure rapid and efficient peptide release eRF3 interacts physically with Pabp • In yeast, eRF3 is also known as Sup35p.

Recycling factors

DOM34 & HBS1 • Resemble eRF1/eRF3 complex, Hbs1 is a GTPase • Facilitate dissociation of 80S ribosomes, required also for No-Go-decay (NGD) peptidyl-tRNA release • In yeast, Hbs1 has a paralog, SKI7 required for No-Stop-Decay (NSD)

ABCE1/RLI1 • Essential Fe-S protein, required together with Doms34 & Hbs1 for 80S dissociation • Facilitates binding of eIF3 / multifactor complex (MFC) to 40S

tRNA mimicry of proteins binding to the A-site of the ribosome

Reference: Y Nakamura & K Ito Trends Biochem. Sci. 28: 99-105, 2003

Polypeptide chain termination

Reference:

eRF3 and GTP hydrolysis

Reference: Y Nakamura & K Ito Trends Biochem. Sci. 28: 99-105, 2003

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

Termination of translation by class I release factors

Reference: Schmeing and Ramakrishnan (2009), Nature 461, 1234-1242

Peptidyl-tRNA hydrolysis

Reference: H Song et al. Cell 100: 311-321, 2000

The ends talk to each other

Figure 6-76a Molecular Biology of the Cell (© Garland Science 2008)

Ribosome recycling

Reference: L Rajkowitsch et al. (2004), J. Mol. Biol. 335: 71-85

Dom34-Hbs1 mimick eRF1-eRF3 binding to ribosomes

Reference: T Becker et al. (2011) Nature Structural & Molecular Biology 18, 715-720

• Dom34-Hbs1 is required for dissociation of non-translating ribosomes and is also involved in NGD & NSD (see

below).

• Yeast Dom34 (Pelota in humans) and the GTPase Hbs1 form a complex structurally similar to eRF1 and eRF3.

• Together with ABCE1/Rli1 they promote the dissociation of ribosomal subunits by binding to the A-site in a

codon –independent manner, followed by GTP hydrolysis, dissociation of Hbs1 and accomodation of Dom34 in the

ribosome. ABCE1/Rli then binds and induces ATP-dependent subunit-dissociation.

• Here: Dom34-Hbs1 at the A-site, the P-site is occupied by a tRNA (green) as compared to eRF1-eRF3-GTP. Upon

GTP hydrolysis, eRF1 (blue) snaps back towards the decoding center.

Recycling of ribosomes is assisted by ABCE1/RLI1

Reference: Schweingruber et al. (2013) BBA 1829, 612-623

Beginning and end of translation are coordinated by eIF3

Reference: Beznoskova et al. (2013) PLOS Genetics 9, e1003906

The Structure of Translationally Silenced Yeast Ribosomes at 3.0 Å

Reference: A Ben-Shem et al. (2011) Science 334, 1524-1529

Translationally Silenced Ribosomes

• Highest resolution structures have been obtained from 80S

ribosomes isolated from yeast cells starved for glucose.

They do not carry mRNA and are translationally silenced.

Under stress conditions, Stm1 maintains 80S ribosomes

coupled and inactive

• The only associated non-ribosomal protein is Stm1, which

interacts with both 40S (blue) and 60S (yellow) subunits

(A).

• The top view of the 40S head and the CP of 60S shows

how Stm1 follows the mRNA pathway until the P-site (B;

dashed black line represents the mRNA).

• Dom34-Hbs1 & Rli1 are required to dissociate inactive

ribosomes that accumulate in glucose-starved yeast (van den

Elzen et al. (2014) EMBO J. 33, 265-276).

Abnormal termination events leading to rapid mRNA degradation

Reference: Roy & Jacobson Trends in Genetics (2013) 29.doi:10.1016

NMD: Nonsense Mediated Decay due to premature stop codon NGD: No-Go Decay due to stalled ribosomes NSD: No-Stop Decay due to lack of a stop codon

Seminar 2

-  Where comes the energy for peptide bond formation from?

-  What are the implications of the finding that peptidyltransferase is a ribozyme for our view

of evolution?

-  Compare polyribosome profiles from cells with inhibited initiation with those of cells with

inhibited elongation!

-  Aminoglycosides: what are they and how could they affect eukaryotic cells?

-  What are the signals that activate or inactivate EF2 kinase?

-  mTor inactivation inhibits both translation initiation and elongation, how?

- What is the GGQ motiv of eRF1, what is its function?

-  What is eIF3, what is its function?