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Probing Editing Domain Conformational Changes Upon E. coli Prolyl-tRNA Synthetase•YbaK Complex Formation

Sackes, Zubeyde
http://rave.ohiolink.edu/etdc/view?acc_num=osu1291022397

Abstract Details

2010, Master of Science, Ohio State University, Chemistry.
Aminoacyl-tRNA synthetases (aaRSs) are multidomain enzymes, which function in protein synthesis by activating cognate amino acids and transferring them to their cognate tRNAs. To achieve high fidelity in protein translation, some synthetases have evolved proofreading functions, and in a few cases, these functions are carried out by free-standing proteins. Lack of editing ability of aaRSs can cause mis-acylation of tRNAs, which results in growth defects in Escherichia coli, and neurodegeneration or an apoptotic response in mammalian cells. We are interested in the interaction between E. coli prolyl-tRNA synthetase (ProRS) and E. coli YbaK during editing. Bacterial ProRSs misactivate Ala and Cys and contain a large insertion (INS) domain between motifs 2 and 3 of the conserved active site, which is responsible for Ala-tRNAPro editing. However, the editing domain of this enzyme is unable to hydrolyze mischarged Cys-tRNAPro. Interestingly, a small free-standing YbaK protein, which is homologous to INS domain, possesses Cys-tRNAPro editing activity and interacts with the ProRS/tRNA complex. Based on the crystal structures of some class I and II synthetases, significant rotation of the editing domain occurs upon substrate binding. The crystal structure of Enteracoccus faecalis ProRS indicates the lack of a strong interaction between the INS domain and the catalytic core. Therefore, we hypothesize that the INS domain is flexibly linked and could rotate during the post-transfer editing mechanism. In this work, we investigated editing domain conformational changes upon E. coli ProRS•YbaK complex formation using biophysical methods. Förster resonance energy transfer (FRET) was used to monitor conformational change in the INS domain upon YbaK binding and vice versa. A donor probe was positioned in the elbow region (TΨC loop position C61) of tRNAPro using a semi-synthetic construction strategy. An acceptor probe was attached to a surface exposed helix in the INS domain by mutation of a A329 to Cys, and on a surface exposed region of YbaK (A124C mutant). Based on time-resolved- FRET data, we observed that ProRS addition to the YbaK•tRNA complex does not significantly perturb the interaction between YbaK and tRNA. In contrast, YbaK addition to a ProRS•tRNAPro complex results in a modest increase in the tRNA-INS distance. Computational docking was used to rationalize the measured distances and distance changes. A plausible explanation for the measured distance between YbaK and tRNA in the absence and presence of ProRS is provided, however, the measured distances between ProRS and tRNA are not consistent with available models. These results suggest either that the position of the INS domain in solution differs substantially from the available cyrstal structure, or that the acceptor probe on ProRS is not freely rotating, thus obscuring the distances measured by FRET. Future studies are needed to resolve this discrepancy.
Karin Musier-Forsyth (Advisor)
Michael Ibba (Committee Member)
99 p.
Chemistry
aminoacyl-tRNA synthetases; prolyl-tRNA synthetase; ProRS&8226; YbaK complex; F&246; rster resonance energy transfer

Recommended Citations

Citations

  • Sackes, Z. (2010). Probing Editing Domain Conformational Changes Upon E. coli Prolyl-tRNA Synthetase•YbaK Complex Formation [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1291022397

    APA Style (7th edition)

  • Sackes, Zubeyde. Probing Editing Domain Conformational Changes Upon E. coli Prolyl-tRNA Synthetase•YbaK Complex Formation. 2010. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1291022397.

    MLA Style (8th edition)

  • Sackes, Zubeyde. "Probing Editing Domain Conformational Changes Upon E. coli Prolyl-tRNA Synthetase•YbaK Complex Formation." Master's thesis, Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1291022397

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.