Holtkamp, Wolf

[["dc.bibliographiccitation.firstpage","1073"],["dc.bibliographiccitation.issue","9"],["dc.bibliographiccitation.journal","The EMBO journal"],["dc.bibliographiccitation.lastpage","1085"],["dc.bibliographiccitation.volume","33"],["dc.contributor.author","Holtkamp, Wolf"],["dc.contributor.author","Cunha, Carlos E. da"],["dc.contributor.author","Peske, Frank"],["dc.contributor.author","Konevega, Andrey L."],["dc.contributor.author","Wintermeyer, Wolfgang"],["dc.contributor.author","Rodnina, Marina V."],["dc.date.accessioned","2018-01-26T06:04:53Z"],["dc.date.available","2018-01-26T06:04:53Z"],["dc.date.issued","2014"],["dc.description.abstract","Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G-GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit."],["dc.identifier.doi","10.1002/embj.201387465"],["dc.identifier.pmid","24614227"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/11834"],["dc.language.iso","en"],["dc.notes.status","final"],["dc.relation.eissn","1460-2075"],["dc.title","GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits"],["dc.type","journal_article"],["dc.type.internalPublication","unknown"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","908"],["dc.bibliographiccitation.issue","10"],["dc.bibliographiccitation.journal","BioEssays"],["dc.bibliographiccitation.lastpage","918"],["dc.bibliographiccitation.volume","36"],["dc.contributor.author","Holtkamp, Wolf"],["dc.contributor.author","Wintermeyer, Wolfgang"],["dc.contributor.author","Rodnina, Marina V."],["dc.date.accessioned","2017-09-07T11:45:30Z"],["dc.date.available","2017-09-07T11:45:30Z"],["dc.date.issued","2014"],["dc.description.abstract","The translocation of tRNAs through the ribosome proceeds through numerous small steps in which tRNAs gradually shift their positions on the small and large ribosomal subunits. The most urgent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase elongation factor G (EF-G), promotes directed movement; and (iii) how the energy of GTP hydrolysis is coupled to movement. In the light of recent advances in biophysical and structural studies, we argue that intermediate states of translocation are snapshots of dynamic fluctuations that guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the energy regimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms."],["dc.identifier.doi","10.1002/bies.201400076"],["dc.identifier.gro","3142042"],["dc.identifier.isi","000342914600002"],["dc.identifier.pmid","25118068"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/3901"],["dc.language.iso","en"],["dc.notes.intern","WoS Import 2017-03-10"],["dc.notes.status","final"],["dc.notes.submitter","PUB_WoS_Import"],["dc.relation.eissn","1521-1878"],["dc.relation.issn","0265-9247"],["dc.title","Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.peerReviewed","yes"],["dc.type.subtype","original"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","e24315"],["dc.bibliographiccitation.issue","1"],["dc.bibliographiccitation.journal","Translation"],["dc.bibliographiccitation.volume","1"],["dc.contributor.author","Cunha, Carlos E."],["dc.contributor.author","Belardinelli, Riccardo"],["dc.contributor.author","Peske, Frank"],["dc.contributor.author","Holtkamp, Wolf"],["dc.contributor.author","Wintermeyer, Wolfgang"],["dc.contributor.author","Rodnina, Marina V."],["dc.date.accessioned","2018-01-26T08:50:21Z"],["dc.date.available","2018-01-26T08:50:21Z"],["dc.date.issued","2013"],["dc.description.abstract","Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor."],["dc.format.extent","1-11"],["dc.identifier.doi","10.4161/trla.24315"],["dc.identifier.pmid","26824016"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/11848"],["dc.language.iso","en"],["dc.notes.status","final"],["dc.title","Dual use of GTP hydrolysis by elongation factor G on the ribosome"],["dc.type","journal_article"],["dc.type.internalPublication","unknown"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","11858"],["dc.bibliographiccitation.issue","20"],["dc.bibliographiccitation.journal","Nucleic acids research"],["dc.bibliographiccitation.lastpage","11866"],["dc.bibliographiccitation.volume","45"],["dc.contributor.author","Mercier, Evan"],["dc.contributor.author","Holtkamp, Wolf"],["dc.contributor.author","Rodnina, Marina V."],["dc.contributor.author","Wintermeyer, Wolfgang"],["dc.date.accessioned","2018-01-17T12:49:58Z"],["dc.date.available","2018-01-17T12:49:58Z"],["dc.date.issued","2017-11-16"],["dc.description.abstract","The bacterial signal recognition particle (SRP) is part of the machinery that targets ribosomes synthesizing membrane proteins to membrane-embedded translocons co-translationally. Recognition of nascent membrane proteins occurs by virtue of a hydrophobic signal-anchor sequence (SAS) contained in the nascent chain, usually at the N terminus. Here we use fluorescence-based stopped-flow to monitor SRP-ribosome interactions with actively translating ribosomes while an SRP substrate is synthesized and emerges from the peptide exit tunnel. The kinetic analysis reveals that, at cellular concentrations of ribosomes and SRP, SRP rapidly binds to translating ribosomes prior to the emergence of an SAS and forms an initial complex that rapidly rearranges to a more stable engaged complex. When the growing peptide reaches a length of ∼50 amino acids and the SAS is partially exposed, SRP undergoes another conformational change which further stabilizes the complex and initiates targeting of the translating ribosome to the translocon. These results provide a reconciled view on the timing of high-affinity targeting complex formation, while emphasizing the existence of preceding SRP recruitment steps under conditions of ongoing translation."],["dc.identifier.doi","10.1093/nar/gkx888"],["dc.identifier.pmid","29149347"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/11688"],["dc.identifier.url","https://sfb1190.med.uni-goettingen.de/production/literature/publications/14"],["dc.language.iso","en"],["dc.notes.status","zu prüfen"],["dc.relation","SFB 1190: Transportmaschinen und Kontaktstellen zellulärer Kompartimente"],["dc.relation","SFB 1190 | P16: Co-translationaler Einbau von Proteinen in die bakterielle Plasmamembran"],["dc.relation.eissn","1362-4962"],["dc.relation.workinggroup","RG Rodnina"],["dc.rights","CC BY-NC 4.0"],["dc.title","Signal recognition particle binds to translating ribosomes before emergence of a signal anchor sequence"],["dc.type","journal_article"],["dc.type.internalPublication","unknown"],["dc.type.subtype","original_ja"],["dc.type.version","published_version"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","1332"],["dc.bibliographiccitation.issue","12"],["dc.bibliographiccitation.journal","Nature structural & molecular biology"],["dc.bibliographiccitation.lastpage","1337"],["dc.bibliographiccitation.volume","19"],["dc.contributor.author","Holtkamp, Wolf"],["dc.contributor.author","Lee, Sejeong"],["dc.contributor.author","Bornemann, Thomas"],["dc.contributor.author","Senyushkina, Tamara"],["dc.contributor.author","Rodnina, Marina V."],["dc.contributor.author","Wintermeyer, Wolfgang"],["dc.date.accessioned","2018-01-29T11:01:49Z"],["dc.date.available","2018-01-29T11:01:49Z"],["dc.date.issued","2012"],["dc.description.abstract","Ribosomes synthesizing inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP) pathway. By rapid kinetic analysis we show that after initial binding to the ribosome, SRP undergoes dynamic fluctuations in search of additional interactions. Non-translating ribosomes, or ribosomes synthesizing non-membrane proteins, do not provide these contacts, allowing SRPs to dissociate rapidly. A nascent peptide in the exit tunnel stabilizes SRPs in a standby state. Binding to the emerging signal-anchor sequence (SAS) of a nascent membrane protein halts the fluctuations of SRP, resulting in complex stabilization and recruitment of the SRP receptor. We propose a kinetic model where SRP rapidly scans all ribosomes until it encounters a ribosome exposing an SAS. Binding to the SAS switches SRP into the targeting mode, in which dissociation is slow and docking of the SRP receptor is accelerated."],["dc.identifier.doi","10.1038/nsmb.2421"],["dc.identifier.pmid","23142984"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/11868"],["dc.language.iso","en"],["dc.notes.status","final"],["dc.relation.eissn","1545-9985"],["dc.title","Dynamic switch of the signal recognition particle from scanning to targeting"],["dc.type","journal_article"],["dc.type.internalPublication","unknown"],["dspace.entity.type","Publication"]]