Effects of sequence variation in the core promoter region of the rnpB gene on transcription initiation and regulation

Abstract

The region between the -10 region and transcription start site of E. coil promoters is known as the discriminator that is responsible for transcription regulation of the stringently controlled promoters. However, the precise role of the region in transcription remains undefined. In this thesis, first, effects of sequence variations of the discriminator region of the rnpB P-l promoter on the transcription initiation rates, the stability of initiation complexes, and the responses by ppGpp were examined, in order to see how the discriminator region was involved in transcription. Each sequence in the discriminator region conferred a different weight to these parameters. These results were compared with the stringent responses of the altered promoters. The variation of stringent response by the sequence alteration in the discriminator region could not be simply explained by the change of either the complex stability or the ppGpp-sensitivity alone. Then, the effect of ppGpp on stability of the initiation complexes was examined, and found that ppGpp destabilized the initiation complexes differently according to the sequence of the discriminator region of promoters. The results suggest that the decrease of the stability of open complexes of stringently-controlled promoters by ppGpp is responsible for generating stringent signals. In this thesis, the important of the sequence in the -35 region as a contributor the stringent signal was assessed by using the mpB promoter as a model promoter. The sequence variation in the -35 region changed the stringent signal. Some variations conferred more-stringent signals on the promoter, while others conferred less-stringent signals. The change to TTGACA that perfectly matches the consensus -35 region caused the most-prominent relieving effect on transcription repression under stringent conditions. However, the spacing between the -35 and -10 regions is also significant because the relieving effect of the TTGACA was offset by the cha...