Author response: Dynamics of ribosomes and release factors during translation termination in E. coli

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long β-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

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Decision letter: Dynamics of ribosomes and release factors during translation termination in E. coli

10.7554/elife.34252.022 ◽

2018 ◽

Keyword(s):

Translation Termination ◽

E Coli ◽

Release Factors

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Dynamics of ribosomes and release factors during translation termination in E. coli

10.1101/243485 ◽

2018 ◽

Author(s):

Sarah Adio ◽

Heena Sharma ◽

Tamara Senyushkina ◽

Prajwal Karki ◽

Cristina Maracci ◽

...

Keyword(s):

Single Molecule ◽

Translation Termination ◽

Bound State ◽

Gtp Hydrolysis ◽

E Coli ◽

Release Factors ◽

Single Molecule Fret ◽

Peptide Release ◽

Two Factors ◽

Stochastic Assembly

AbstractRelease factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

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Methylation of E. coli Class I Release Factors Increases the Accuracy of Translation Termination in vitro

10.1101/239822 ◽

2018 ◽

Author(s):

Gürkan Korkmaz

Keyword(s):

Translation Termination ◽

Class I ◽

Release Factor ◽

E Coli ◽

Release Factors ◽

Peptide Release ◽

Ribosomal Protein Synthesis ◽

Uncompetitive Inhibitor ◽

Menten Constant

ABSTRACTRibosomal protein synthesis (translation) is a highly accurate process. Translation termination, in particular, must be accurate to prevent truncated proteins. How this accuracy is achieved is not fully understood in all its details. Using an E. coli in vitro system, I explore novel mechanisms that contribute to the high accuracy of translation termination. By comparing the Michaelis-Menten parameters of methylated and non-methylated release factors on cognate and non-cognate codons. Post-translational methylation of a strictly conserved GGQ motif in class I release factors increases the accuracy of termination by up to 5-fold. This happens by increasing both the maximum rate of peptide release (kcat) and Michaelis-Menten constant (KM). Further, I demonstrate here that a non-methylated release factor acts like an uncompetitive inhibitor of enzyme reactions. Overall, this study shows that the methylation of class I release factors is a novel mechanism contributing to highly accurate translation termination.AbbreviationsRFrelease factorRCrelease complex

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Extensive Ribosome and RF2 Rearrangements during Translation Termination

10.1101/600445 ◽

2019 ◽

Author(s):

Egor Svidritskiy ◽

Gabriel Demo ◽

Anna B. Loveland ◽

Chen Xu ◽

Andrei A. Korostelev

Keyword(s):

Protein Synthesis ◽

Stop Codon ◽

Translation Termination ◽

Single Sample ◽

Peptidyl Transferase ◽

E Coli ◽

Peptidyl Transferase Center ◽

Release Factors ◽

Subunit Rotation ◽

Intersubunit Rotation

AbstractProtein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long β-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

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Dynamics of ribosomes and release factors during translation termination in E. coli

eLife ◽

10.7554/elife.34252 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 19

Author(s):

Sarah Adio ◽

Heena Sharma ◽

Tamara Senyushkina ◽

Prajwal Karki ◽

Cristina Maracci ◽

...

Keyword(s):

Single Molecule ◽

Translation Termination ◽

Bound State ◽

Gtp Hydrolysis ◽

E Coli ◽

Release Factors ◽

Single Molecule Fret ◽

Biochemical Assays ◽

Peptide Release ◽

Two Factors

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

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