Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Specific ribonucleotides within the 5{dollar}\sp\prime{dollar} domain of 16 S rRNA were altered by deletion and/or substitution by site-directed mutagenesis of cloned DNA to assess the importance of these particular residues in defining the binding site for ribosomal protein S20. Gel filtration and sucrose gradient centrifugation were employed to measure the binding of S20 to synthetic transcripts containing various alterations. RNA transcripts containing residues 1-402 were sufficient for optimal S20 binding. The removal or the substitution of bulges at 250-251, 278-280 or 321/332 reduced S20-16 S rRNA association significantly. A variety of mutations was introduced into the stem and loop residues of a hairpin structure spanning residues 316-337. These changes demonstrated a strong requirement for both the specific A321*G332 bulge and for residues in the loop itself for proper S20 binding. The nucleotides identified as crucial for S20 binding probably constitute direct contacts between 16 S rRNA and S20.;Processing of 9 S precursor RNA requires the endoribonuclease RNase E which makes two cleavages to liberate p5, the immature form of 5 S rRNA. The contributions of primary and secondary structure to RNase E-mediated cleavage of 9 S RNA were investigated. Site-directed mutagenesis of a cloned 9 S RNA sequence was performed and synthetic transcripts derived from a variety of such mutant templates were assayed as substrates for RNase E-dependent endonuclease activity in fractionated extracts. Our results suggest that RNase E specifically recognizes and cleaves single-stranded RNA sequences only when presented in a proper conformational context. Adjacent secondary structures appear to play a direct and critical role in the enzyme's recognition of its substrate. Additionally, it may serve to anchor single-stranded regions to ensure the availability of the RNase E cleavage sites.;The role of the polypeptide encoded by ams/rne/hmp1, a gene required for RNase E activity, was investigated. Northwestern blotting and UV crosslinking of substrate RNA to crude fractions containing RNase E unambiguously showed that overexpressed Ams/Rne/Hmp1 polypeptide has a high affinity for RNA. Our results demonstrate that the ams/rne/hmp1 gene product intimately associates with RNase E substrate RNA and supports the notion that Ams/Rne/Hmp1 is the nuclease responsible for RNase E activity. In addition, the RNA-binding domain of Ams/Rne/Hmp1 was localized and a putative RNase E-associated protein was identified.



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