The experiments in this proposal are aimed at understanding aspects of mRNA decay and mRNA processing in the Gram-positive bacterium Bacillus subtilis. Rapid turnover of bacterial mRNA is an important element in regulating gene expression, and allows quick adaptation of microorganisms to changing environmental conditions. Decay is thought to initiate with an endonucleolytic cleavage in the body of the message, followed by 3'-5' exonucleolytic degradation. The gene for polynucleotide phosphorylase (PNPase), a major 3' -to- 5' exoribonuclease of B. Subtilis, has recently been cloned, and a strain that contains a deletion of this gene (the pnpA deletion strain) has been constructed. The absence of the major 3'-to-5 exoribonuclease in the pnpA deletion strain has allowed detection of intermediates in the mRNA decay process. We propose to characterize these decay intermediates, with the expectation that this will provide new information about the sites at which initiation of decay occurs. Furthermore, the absence of PNPase in this strain has revealed the significance of an alternative 3'- to-5' exonuclease, which is Mn2+ dependent, and cloning of the gene for this ribonuclease is proposed. Finally, the PNPase deficient strain has several mutant phenotypes, most notably sensitivity to growth in the presence of tetracycline. We hypothesize that these mutant phenotypes are due to altered expression by mRNA processing. Another recently cloned B. Subtilis gene codes for Bs-RNase III, a narrow-specificity endoribonuclease. We propose to construct strains that are deficient in Bs-RNase III in order to study the function of this enzyme in RNA processing and gene expression. Other endoribonuclease activities are likely to be present in B. Subtilis, and an in vitro system will be set up to detect such activities.
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