Date of Award
1988
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
The ribosomal protein S20 of Escherichia coli is one of only six small subunit proteins capable of binding independently to 16S rRNA. To define the regions of S20 required for binding to 16S rRNA, the binding of S20 and a number of C-terminal deletion mutants to 16S rRNA were measured. Mutant S20s constructed using recombinant DNA technology were synthesized in vitro by coupled transcription-translation and assayed directly. The affinity of S20 produced in vitro for 16S rRNA was found to be 1.2 {dollar}\times{dollar} 10{dollar}\sp7{dollar} M{dollar}\sp{lcub}-1{rcub}{dollar} using a gel filtration assay. Removal of just 6 residues from the C-terminus of the protein resulted in a sharp drop in binding activity, suggesting the presence of critical residues in this region.;To characterize the functional basis for the ability of S20 to regulate its own production at a post-transcriptional step, the affinity of S20 for S20 mRNA was also measured. RNA transcripts of the S20 operon were generated in vitro and affinity for S20 was measured by three methods: gel filtration, filter binding, and gel retardation. Only gel filtration was capable of clearly measuring binding of S20 to a positive control consisting of residues 1 to 560 of 16S rRNA transcribed in vitro. When S20 mRNA was utilized in this assay no S20 binding was detectable, indicating the K{dollar}\sb{lcub}\rm a{rcub}{dollar} of S20 for its own messenger is probably no greater than about 10{dollar}\sp5{dollar} M{dollar}\sp{lcub}-1{rcub}{dollar}. This suggests the possibility that S20 may interact with more complex intermediates involved in the initiation of its own translation, rather than with mRNA alone.;Finally, the roles of the S20 leader and UUG initiation codon in autoregulation and translation efficiency were also examined. Deletions and point mutations in these sequences were tested for efficiency of S20 translation in vitro. A change in the initiation codon from UUG to AUG was found to cause a significant increase in translational efficiency as well as almost complete relief from regulation. The intrinsic translational efficiency of the S20 mRNA is apparently one of the primary determinants of the extent of autoregulation in S20 expression.
Recommended Citation
Donly, Brooke Cameron, "Structure-function Relationships In The Rna Binding Activities Of Ribosomal Protein S20 Of Escherichia Coli" (1988). Digitized Theses. 1740.
https://ir.lib.uwo.ca/digitizedtheses/1740