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

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.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
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.

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

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

scholarly journals N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

2021 ◽  
Vol 49 (5) ◽  
pp. 2684-2699
Author(s):  
Ka-Weng Ieong ◽  
Gabriele Indrisiunaite ◽  
Arjun Prabhakar ◽  
Joseph D Puglisi ◽  
Måns Ehrenberg
Keyword(s):  
Single Molecule ◽  
Stop Codon ◽  
Product Formation ◽  
Release Factors ◽  
Substrate Complex ◽  
Peptidyl Transfer ◽  
Enzyme Substrate ◽  
Peptide Release ◽  
Accuracy Ratio ◽  
Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

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

2018 ◽  
Author(s):  
Sarah Adio ◽  
Heena Sharma ◽  
Tamara Senyushkina ◽  
Prajwal Karki ◽  
Cristina Maracci ◽  
...  
Keyword(s):  
Translation Termination ◽  
Author Response ◽  
Response Dynamics ◽  
E Coli ◽  
Release Factors

scholarly journals Mechanistic basis for motor-driven membrane constriction by dynamin

2020 ◽  
Author(s):  
Oleg Ganichkin ◽  
Renee Vancraenenbroeck ◽  
Gabriel Rosenblum ◽  
Hagen Hofmann ◽  
Alexander S. Mikhailov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Model ◽  
Molecular Motor ◽  
Coarse Grained ◽  
Gtp Hydrolysis ◽  
Single Molecule Fret ◽  
Membrane Tube ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Gtpase Cycle

AbstractThe mechano-chemical GTPase dynamin assembles on membrane necks of clathrin-coated vesicles into helical oligomers that constrict and eventually cleave the necks in a GTP-dependent way. It remains not clear whether dynamin achieves this via molecular motor activity and, if so, by what mechanism. Here, we used ensemble kinetics, single-molecule FRET and molecular dynamics simulations to characterize dynamin’s GTPase cycle and determine the powerstroke strength. The results were incorporated into a coarse-grained structural model of dynamin filaments on realistic membrane templates. Working asynchronously, dynamin’s motor modules were found to collectively constrict a membrane tube. Force is generated by motor dimers linking adjacent helical turns and constriction is accelerated by their strain-dependent dissociation. Consistent with experiments, less than a second is needed to constrict a membrane tube to the hemi-fission radius. Thus, a membrane remodeling mechanism relying on cooperation of molecular ratchet motors driven by GTP hydrolysis has been revealed.

scholarly journals The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex

2020 ◽  
Vol 295 (51) ◽  
pp. 17646-17658
Author(s):  
Fang-Yuan Teng ◽  
Ting-Ting Wang ◽  
Hai-Lei Guo ◽  
Ben-Ge Xin ◽  
Bo Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Premature Aging ◽  
Binding Modes ◽  
Ssdna Binding ◽  
Recq Helicases ◽  
E Coli ◽  
Single Molecule Fret ◽  
G4 Dna ◽  
Long Time

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

scholarly journals Inhibition of translation termination by small molecules targeting ribosomal release factors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xueliang Ge ◽  
Ana Oliveira ◽  
Karin Hjort ◽  
Terese Bergfors ◽  
Hugo Gutiérrez-de-Terán ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Small Molecules ◽  
Translation Termination ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Release Factors ◽  
Bacterial Ribosome ◽  
Peptide Release ◽  
Bacterial Protein Synthesis ◽  
Important Drug

Abstract The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

scholarly journals Extensive ribosome and RF2 rearrangements during translation termination

eLife ◽  
2019 ◽  
Vol 8 ◽  
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

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.

scholarly journals Spotlighting motors and controls of single FoF1-ATP synthase

2013 ◽  
Vol 41 (5) ◽  
pp. 1219-1226 ◽  
Author(s):  
Michael Börsch ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Structural Information ◽  
E Coli ◽  
Single Molecule Fret ◽  
Membrane Enzyme ◽  
Subunit Rotation ◽  
Fof1 Atp Synthase

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.

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