Analysis of Rnase P RNA Structure and Function

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Title: Analysis of Rnase P RNA Structure and Function
Author: Willams, Daniel
Advisors: Dr. James W. Brown, Chair
Dr. Amy Grunden, Member
Dr. Cynthia Hemenway, Member
Dr. Paul Bishop, Member
Abstract: The diversity of Archaea in municipal wastewater sludge was investigated by amplification of rRNA sequences from sludge DNA using archaeal-specific primers. The most common sequences were extreme halophiles; also found were sequence members of environmental euryarchaeal and crenarchaeal groups. Only distant relatives of Methanosarcina among the Methanomicrobiales were found. A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P have largely disappeared, and even variation in the sequence and structure in the RNAs are similar in extent and type. The structure of the cruciform (P7-P11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences. Archaeal and bacterial RNase P RNAs are very similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions. In order to assess how readily the activity of the archaea RNA alone could be improved by point mutations in its sequence, in vitro selection was used to generate variants of the self-cleaving conjugant Methanobacterium formicicum: B. subtilus tRNAAsp (cpTP). Functional variants were generated with a broad spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection were only comparable to wildtype in catalytic activity; more performed significantly faster at lower ionic strength. These results suggest that the archaeal RNase P RNA is globally optimized and that the protein may play larger compensatory roles in catalysis than previously thought.
Date: 2001-08-21
Degree: PhD
Discipline: Microbiology

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