Post-transcriptional Regulation of Gene Expression

The mechanism of RNA processing, and particularly messenger RNA decay, in the Gram-positive bacterium Bacillus subtilis is the major focus of our laboratory. Previous studies concentrated on an inducibly stable mRNA – the mRNA encoded by the erythromycin-resistance gene, ermC . Numerous derivatives of ermC mRNA have been constructed, and these remain useful tools to probe the mechanism of mRNA decay. We have shown that the 5' end of a message plays an important role in determining mRNA half-life, and are currently looking at specific sites on the mRNA at which decay initiates. Several genes encoding ribonucleases have been cloned, including four genes encoding 3'-to-5' exonucleases and one gene encoding a narrow-specificity endonuclease (called "Bs-RNase III"). In order to understand the function of these exonucleases, we are analyzing decay of endogenous mRNAs in mutant strains lacking one or more of these genes. We are concentrating on small, monocistronic mRNAs, as these are amenable to detailed analysis and likely provide few sights for initiation of decay.

Functional homologues for three of the major players in E. coli mRNA decay – RNase E, RNase II, and oligoribonuclease – have so far not been identified in B. subtilis. A combination of genetic and biochemical approaches is being pursued to identify new B. subtilis genes that are involved in RNA processing. A major goal is to discover the 5'-end-dependent endonuclease that is believed to initiate mRNA decay. Another major goal is to identify other exoribonuclease activities, since a mutant strain missing all four of the currently known exonucleases is viable and clearly contains one or more other 3'-to-5' exonuclease activities.

Ongoing projects in the lab and in collaboration with other laboratories, include:

• the effect of polyadenylation on mRNA decay. We have demonstrated that B. subtilis mRNA is polyadenylated, but the identity of the B. subtilis poly(A) polymerase has been a mystery. Biochemical purification of a poly(A) polymerase activity is anticipated, which will be followed by genetic studies of a strain lacking this activity.
• endoribonuclease cleavage at a site of ribosome stalling. We have demonstrated that such cleavage occurs but have so far not identified the gene specifying this activity.
• basis for essential nature of Bs-RNase III. Strains lacking this activity are generally non-viable, but the basis for this is not known. A rare strain that is missing Bs-RNase III has been recovered, and this strain presumably contains a secondary mutation that compensates for the missing crucial Bs-RNase III activity.