Measuring the Mechanical Forces of Elongation Factor G Mediated Translocation

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dc.contributor.advisorWang, Yuhong
dc.contributor.committeeMemberBriggs, James M.
dc.contributor.committeeMemberDo, Loi H.
dc.contributor.committeeMemberGunaratne, Preethi H.
dc.creatorGavriliuc, Miriam Nicolette
dc.creator.orcid0000-0001-9812-5888
dc.date.accessioned2023-01-14T23:40:41Z
dc.date.createdMay 2022
dc.date.issued2022-04-14
dc.date.updated2023-01-14T23:40:42Z
dc.description.abstractThe ribosome is the ribonucleoprotein complex that is responsible for the correct translation of mRNA into protein. This complex is associated with the GTPase elongation factor G (EF-G), which catalyzes translocation on the ribosome, however, the mechanism is poorly understood. Crystallographic studies revealed a previously unknown compact conformation of EF-G, implying large conformational changes. The biological relevance of this change was not revealed, however, because of the artificial crystal lattice present in x-ray studies. To explore this, an EF-G mutant carrying only two cysteines (M5 EF-G) was generated and internally crosslinked to itself, using two lengths of crosslinker. One that restricted EF-G from fully extending (BM(PEG)6) and one that allowed EF-G to undergo full conformational changes (BM(PEG)11). BM(PEG)11 crosslinked EF-G functioned comparably to wild type, while BM(PEG)6 crosslinked EF-G resulted in a lower force exerted. The BM(PEG)6 crosslinker also reduced ribosomal translocation efficiency to half, compared to that of the longer crosslinker and non-crosslinked EF-G. This force reduction did not result in frameshifting, confirming that EF-G does exert a force, but does not contribute to reading frame maintenance. Studying the overall conformational changes was important for understanding EF-G mechanism, however, it was also important to observe the details leading to the full conformational changes. In a second exploration of EF-G, the effector loop region was studied. The effector loop region spans approximately 7 residues within the GTPase center, the region where GTP is bound and hydrolyzed, on domain I of EF-G and contains important interactions for the function of EF-G. By introducing single site substitutions in the effector loop, the role of each residue was examined. Six variants were produced, and of the six, one was completely unable to hydrolyze GTP, while another exhibited reduced GTPase activity. Not a single of the six was able to promote translocation at a rate comparable to M5 EF-G, effectively abolishing the function of EF-G on the ribosome. By studying the effector loop of EF-G, further understanding of the roles of the residues within this region, and how they contribute to the overall function of EF-G was obtained.
dc.description.departmentBiology and Biochemistry, Department of
dc.format.digitalOriginborn digital
dc.format.mimetypeapplication/pdf
dc.identifier.citationPortions of this document appear in: Yin, H., Gavriliuc, M., Lin, R., Xu, S., & Wang, Y. (2019). Modulation and Visualization of EF‐G Power Stroke During Ribosomal Translocation. Chembiochem, 20(23), 2927-2935.
dc.identifier.urihttps://hdl.handle.net/10657/13322
dc.language.isoeng
dc.rightsThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).
dc.subjectEF-G
dc.subjectRibosome
dc.subjectTranslocation
dc.titleMeasuring the Mechanical Forces of Elongation Factor G Mediated Translocation
dc.type.dcmiText
dc.type.genreThesis
dcterms.accessRightsThe full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period.
local.embargo.lift2024-05-01
local.embargo.terms2024-05-01
thesis.degree.collegeCollege of Natural Sciences and Mathematics
thesis.degree.departmentBiology and Biochemistry, Department of
thesis.degree.disciplineBiochemistry
thesis.degree.grantorUniversity of Houston
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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