Identification of a biological role for the Escherichia coli small stable RNA, 10Sa RNA.

Abstract

This work addresses the role of the Escherichia coli ssrA gene product, 10Sa RNA, a small, stable, relatively abundant RNA. This RNA is conserved among diverse genera. The effect of ssrA mutations on the growth of bacteriophage $\lambda$, hybrid phage formed between $\lambda$ and P22, as well as the expression of the E. coli lac operon is examined. Certain $\lambda$-P22 hybrids, providing they express the P22 C1 protein, fail to grow in E. coli with the sipB391 mutation. The sipB39l mutation is shown to be a large deletion that extends into the 3\sp\prime end of the ssrA gene. This deletion leaves most of ssrA intact, but removes the sequence encoding the 3\sp\prime end of the precursor form of 10Sa RNA. The lack of functional 10Sa RNA appears to be responsible for the inhibition of $\lambda$-P22 growth in E. coli with the sipB391 mutation. Altering the P22 DNA binding protein C1 so that it binds DNA less tightly alleviates the effect of the ssrA mutation. A single base pair mutation in $\lambda imm$P22 that changes both the DNA target of C1, the $-$35 region of the P\sb{\rm RE} promoter, and the amino terminus of the C1 protein allows for phage growth in an ssrA mutant. Although the mutant C1 protein activates the mutant P\sb{\rm RE} promoter, it fails to activate the wild type P\sb{\rm RE} promoter, suggesting that a single base pair mutation changes the specificities of both a protein and its target site. We propose that the inhibition of $\lambda imm$P22 growth is due to a more avid binding of C1 to its target in the absence of 10Sa RNA. The avidity of the phage $\lambda$ cI and E. coli LacI repressors for their cognate operator sites is also increased in E. coli ssrA mutants. Moreover, these repressor proteins bind to 10Sa RNA in vitro, even in the presence of a 100 fold excess of tRNA. These results suggest that RNAs may contribute to regulation of gene expression by modulating protein-DNA interactions.Ph.D.Microbiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104534/1/9527728.pdfDescription of 9527728.pdf : Restricted to UM users only