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Substrate specific contributions of the protein subunit of Escherichia coli RNase P to substrate recognition and catalysis.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Substrate specific contributions of the protein subunit of Escherichia coli RNase P to substrate recognition and catalysis./
Author:	Sun, Lei.
Description:	158 p.
Notes:	Adviser: Michael E. Harris.
Contained By:	Dissertation Abstracts International68-08B.
Subject:	Biology, Molecular. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3276742
ISBN:	9780549180043

Substrate specific contributions of the protein subunit of Escherichia coli RNase P to substrate recognition and catalysis.
Sun, Lei.

Substrate specific contributions of the protein subunit of Escherichia coli RNase P to substrate recognition and catalysis. - 158 p.

Adviser: Michael E. Harris.

Thesis (Ph.D.)--Case Western Reserve University, 2008.

Keywords. RNase P, C5 protein, substrate recognition, catalysis

ISBN: 9780549180043Subjects--Topical Terms:

1017719
Biology, Molecular.

Substrate specific contributions of the protein subunit of Escherichia coli RNase P to substrate recognition and catalysis.
LDR:03477nam 2200301 a 45 001 852049
005 20100629
008 100629s2008 ||||||||||||||||| ||eng d
020 $a 9780549180043
035 $a (UMI)AAI3276742
035 $a AAI3276742
040 $a UMI $c UMI
100 1 $a Sun, Lei. $3 1017718
245 1 0 $a Substrate specific contributions of the protein subunit of Escherichia coli RNase P to substrate recognition and catalysis.
300 $a 158 p.
500 $a Adviser: Michael E. Harris.
500 $a Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5022.
502 $a Thesis (Ph.D.)--Case Western Reserve University, 2008.
520 $a Keywords. RNase P, C5 protein, substrate recognition, catalysis
520 $a E. coli RNase P is an endonuclease that catalyzes the maturation of tRNA in cells. It consists of a &sim;400 nucleotide RNA subunit (P RNA) and a &sim;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.
520 $a 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 Mg 2+ 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.
520 $a 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-tRNAMet605 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.
590 $a School code: 0042.
650 4 $a Biology, Molecular. $3 1017719
690 $a 0307
710 2 $a Case Western Reserve University. $3 1017714
773 0 $t Dissertation Abstracts International $g 68-08B.
790 $a 0042
790 1 0 $a Harris, Michael E., $e advisor
791 $a Ph.D.
792 $a 2008
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3276742