Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


case1188406954.pdf (2.05 MB)

ETD Abstract Container

Abstract Header

SUBSTRATE SPECIFIC CONTRIBUTIONS OF THE PROTEIN SUBUNIT OF E.COLI RNASE P TO SUBSTRATE RECOGNITION AND CATALYSIS

SUN, LEI
http://rave.ohiolink.edu/etdc/view?acc_num=case1188406954

Abstract Details

2008, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
E.coli RNase P is an endonuclease that catalyzes the maturation of tRNA in cells. It consists of a ~400 nucleotide RNA subunit (P RNA) and a ~100 amino acid protein subunit (C5). RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus sequence elements for recognition. To examine how the structural variation in different pre-tRNAs may affect RNase P processing, we compared binding affinities and cleavage rates of E.coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. Remarkably, the binding constants and catalytic rates of different pre-tRNAs are essentially uniform for RNase P holoenzyme but not for P RNA alone. These data suggest that an important biological function of the RNase P protein is to offset the effects of the variation in pre-tRNA structure such that binding and catalysis are uniform. Comparative analyses of pre-tRNA and tRNA binding reveal that the uniform binding results from variations in the energetic contribution of the 5’ leader which serves to compensate for the difference in tRNA binding. Kinetic analyses show that the protein subunit makes a dramatic (>900-fold) contribution to the catalysis of some non-consensus pre-tRNAs. To explore the mechanism of catalytic enhancement by C5 protein, pH dependence and Mg2+ titration experiments were performed. The results from pH dependence indicate that both holoenzyme and P RNA alone reaction have the same rate limiting step which is the chemical cleavage step. And the Mg2+ titration experiments reveal that the catalytic contribution of the protein subunit is primarily due to increasing the affinity of Mg2+ ion binding. Furthermore, to probe the binding of metal ions coordinated to the reactive phosphate, we used a quantitative analysis of Cd2+ rescue of an Rp phosphorothioate modification at the cleavage site. The results demonstrate that RNase P holoenzyme has a tighter binding affinity to the catalytic metal ions than P RNA alone. In addition, to explore the relative importance of the catalytic determinants in pre-tRNA, we performed a systematic structure swapping between a typical consensus E.coli pre-tRNAMet608 and a non-consensus pre-tRNA Met605 to identify the dominant catalytic determinants. It is shown that the primary substrate structure defect in pre-tRNA that needs to be overcome by the protein subunit is the absence of a G+1/C+72 basepair at the cleavage site.
Michael Harris (Advisor)
155 p.
Biology, Molecular
RNase P; C5 protein; substrate recognition; catalysis

Recommended Citations

Citations

  • SUN, L. (2008). SUBSTRATE SPECIFIC CONTRIBUTIONS OF THE PROTEIN SUBUNIT OF E.COLI RNASE P TO SUBSTRATE RECOGNITION AND CATALYSIS [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1188406954

    APA Style (7th edition)

  • SUN, LEI. SUBSTRATE SPECIFIC CONTRIBUTIONS OF THE PROTEIN SUBUNIT OF E.COLI RNASE P TO SUBSTRATE RECOGNITION AND CATALYSIS. 2008. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1188406954.

    MLA Style (8th edition)

  • SUN, LEI. "SUBSTRATE SPECIFIC CONTRIBUTIONS OF THE PROTEIN SUBUNIT OF E.COLI RNASE P TO SUBSTRATE RECOGNITION AND CATALYSIS." Doctoral dissertation, Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1188406954

    Chicago Manual of Style (17th edition)

case1188406954
499
© 2007, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.