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Structure-function relationships in the protein subunit of bacterial ribonuclease P

Jovanovic, Milan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1092422670

Abstract Details

2004, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
Ribonuclease P (RNase P) is a ribonucleoprotein involved in tRNA biosynthesis in all living organisms. Bacterial RNase P is comprised of a catalytic RNA subunit and a lone protein cofactor which plays a supporting, albeit essential, role in the tRNA processing reaction in vivo. In this study, we have searched various databases to identify homologs of the protein subunit of RNase P from diverse bacteria and generated an alignment of their primary sequences to determine the most highly conserved residues. Such an approach has helped us to extend earlier predictions of which residues might play an important role in RNase P catalysis. The amino acid residues identified as important for RNase P catalysis could be categorized in three groups: (i) the RNR motif in helix alpha 2, (ii) the substrate binding cleft, and (iii) amino acid residues involved in the overall stability of the bacterial RNase P protein subunit. By employing site-directed mutagenesis and a genetic complementation assay, we have also gained insights into structure-function relationships in the protein subunit of bacterial RNase P. Specifically, we were able to demonstrate that the bacterial RNase P protein uses one domain to recognize its cognate catalytic RNA subunit and another domain to recognize the 5' leader sequences of its precursor tRNA substrates. The plasticity of the substrate-binding cleft has also been demonstrated by both chemical and genetic rescue experiments. These results, taken together with earlier kinetic studies, have enabled us to understand how the bacterial RNase P protein subunit is able to enhance the rate of chemical cleavage 10-fold and enhance substrate binding 10,000-fold over the RNA alone ptRNA-processing reaction. Finally, we report an interesting study that demonstrates how the Escherichia coli RNase P protein subunit lacking a metal affinity tag can be purified under denaturing conditions using immobilized metal affinity chromatography (IMAC). We are not aware of a precedent in this regard and report these results as a potential new method for the purification of a protein lacking metal affinity tags under denaturing conditions using IMAC.
Venkat Gopalan (Advisor)
305 p.
Chemistry, Biochemistry
Bacterial RNase; RNase; C5 protein; C5; PROTEIN; F18A/F22A; PROTEIN SUBUNIT

Recommended Citations

Citations

  • Jovanovic, M. (2004). Structure-function relationships in the protein subunit of bacterial ribonuclease P [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1092422670

    APA Style (7th edition)

  • Jovanovic, Milan. Structure-function relationships in the protein subunit of bacterial ribonuclease P. 2004. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1092422670.

    MLA Style (8th edition)

  • Jovanovic, Milan. "Structure-function relationships in the protein subunit of bacterial ribonuclease P." Doctoral dissertation, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1092422670

    Chicago Manual of Style (17th edition)

osu1092422670
589
© 2004, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.