Functional analysis of 6S RNA promoters in escherichia coli

Abstract

Small noncoding RNAs (sRNAs) are RNAs ranging from 70 to 500 nt in size, with no protein-coding potential and the major role of sRNAs is regulation of gene expression. Among more than 100 sRNAs of E. coli, 6S RNA is the first sRNA to be sequenced. 6S RNA is transcripted from ssrS gene and interacts specifically with $σ^{70}-RNA$ polymerase (RNAP) to prevent it from associating with its typical target promoter DNA, and then inhibits transcription of the DNA. To understand right-timing expression of $σ^{70}-dependent$ genes in the cell, it is essential to examine how biogenesis of 6S RNA is regulated. 6S RNA can be transcribed from two alternative ssrS promoters, a proximal $σ^{70}-dependent$ promoter, P1, and a distal both $σ^{s}-$ and $σ^{70}-dependent$ promoter, P2. Transcrtips from both promoters should be processed at their 5’ ends to generate mature 6S RNA. However, it is not known yet how transcriptions from two ssrS promoters are coordinately regulated for 6S RNA biogenesis. In this thesis work, first, I attempted to construct ssrS promoter knockout strains to see how each promoter contributes to 6S RNA biogenesis. A P2 knockout strain was obtained, while no P1 knockout strain was formed although the experiments were repeated five times. This result implies that transcription from P1 is essential for cell survival. The P2 knockout strain showed the 6S RNA levels similar to the wild type levels in all growth phases, suggesting that the P2 promoter is not critical to maintenance of the cellular level of 6S RNA. To see whether there is a feedback regulation in ssrS expression or not, I examined changes of transcription from the chromosomal ssrS when P1 or P2 transcripts were overexpressed. I found that the chromosomal ssrS transcription was significantly reduced by multicopy plasmids carrying ssrS gene even if the -10 region of each promoter was mutated. This result allows me to propose a model that a transcription factor, which binds to the upstream promote...