Adaptive mutations in RNA-based regulatory mechanisms: Computational and experimental investigations

Recent discoveries of RNA-based regulatory mechanisms have prompted substantial interest in how they formed and the extent to which varying environmental conditions have influenced their evolution. One class of RNA-based regulatory mechanism that has been found in bacteria is the riboswitch, regulating the biosynthesis of certain vitamins by an RNA genetic control clement that senses small molecules and responds with a structural change that affects transcription termination or translation initiation without the participation of proteins. By taking the thiamin pyrophosphate (TPP)-riboswitch in Bacillus subtilis as a model system, we wish to examine whether beneficial mutations may exist at the level of RNA that will cause an improvement in organism fitness. By computationally analyzing the difference in primary and secondary structure of the B. subtilis TPP-riboswitch collected from the xeric African south-facing slope (SFS) vs. the mesic, European, north-facing slope (NFS) in Evolution Canyon III at Nahal Shaharut, southern Israel, we wish to experimentally study the environmental effect on transcription termination in these RNA-based regulatory mechanisms that are believed to be of ancient origin in the evolutionary time scale. Computational results, so far, indicate that specific mutations affect the riboswitch conformation by causing a global rearrangement. We would like to check whether such mutations could be adaptive mutations that may have a beneficial fitness effect, taking the TPP-riboswitch as a model system at the micro-scale. Empirical results so far indicate that in the promoter region of the TPP-riboswitch, all mutations increase nucleotide GC content in the xeric SFS, whereas in the mesic NFS they increase AT content. Preliminary examination of termination efficiency of strains found exclusively on one slope or the other, reveal increased termination efficiency in the presence of TPP and at more moderate temperatures, but only a suggestion of greater termination efficiency from strains found on both slopes. We expect that further results will shed light on the mutational differences of TPP-riboswitch sequences found on opposite slopes of Evolution Canyon III at Nahal Shaharut, potentially leading to interesting discoveries that relate to the topic of adaptive, nonrandom mutations.