Stability determinants and decay of Bacillus subtilis mRNAs

Sammanfattning: Degradation of mRNA is a controlled process which is as important for gene regulation as the synthesis of mRNA. This thesis focuses on mechanisms of mRNA stability and degradation in Bacillus subtilis, the model of Gram-positive bacteria. The aprE mRNA, encoding an exported protease named subtilisin, is extremely stable with a half-life of more than 25 minutes. Average mRNA half-lives in B. subtilis are around 5 minutes. An aprE leader-lacZ mRNA is as stable as the native aprE mRNA, demonstrating that the aprE leader determines the extreme stability. Mutations were introduced into different domains of the aprE leader and measurements of the half-lives of the corresponding mRNAs showed that a 5’ stem-loop and ribosome loading are responsible for the extreme stability. The Gram-negative Escherichia coli is the bacterium where mRNA degradation is best known. We introduced the aprE leader-lacZ mRNA into E. coli to compare mRNA stability determinants in this bacterium and B. subtilis. The aprE leader mRNA has only average stability in E. coli, demonstrating that the two bacterial species have different mechanisms for mRNA degradation. The half-life of the mRNA is determined by RNase E, the major mRNA-degrading enzyme of E.coli. Moreover, a cleavage in the aprE leader mRNA that was observed in E. coli was not present in B. subtilis. This cleavage is performed by RNase G, an endonuclease homologous to the N-terminal domain of RNase E. Our findings are consistent with the fact that B. subtilis and E. coli have different arsenals of mRNA-degrading enzymes. Finally, we have employed microarrays to measure the stability of a large number of mRNAs in B. subtilis. In total, the stability of about 1500 mRNAs from bacteria in early stationary phase was estimated . This data set made it possible to do comparative analyses of stability determinants. However, so far we have not been able to find any elements that are unique for mRNAs belonging to a certain stability group.