CONSTRUCTION AND ANALYSIS OF ESCHERICHIA COLI HYBRID AND VARIANT PROMOTERS
RUSSELL, DAVID RAY
Doctor of Philosophy
This research has investigated the initiation of transcription in E. coli. The main focus has been the assessment of the effects that specific promoter sequences have on RNA polymerase-promoter interactions, both in vivo and in vitro. Previous studies have shown the importance of two partially conserved hexanucleotide sequences near -10 and -35 of the promoter. However, the specific roles of these sequences, constraints on the locations of these sequences, and contributions from other promoter regions have not been determined. The studies described here have addressed several of these questions. These include: characterization in vitro parameters of the (beta)-lactamase promoter, studies utilizing a variant of the trp promoter, construction of hybrid promoters with different -10 and -35 regions, construction of promoters with altered spacing between the -10 and -35 regions, and construction of promoter variants where adjacent DNA sequences have been altered. The hybrid promoters suggest the spacing between the -35 and -10 regions must be 16 to 18-bp with 17-bp the optimum spacing. The -35 and -10 regions can be used interchangeably and promoter strength increases as the sequence homology of each region approaches the consensus sequence. These data suggest the back half of the trp promoter is several fold stronger than either lac or tet, while the front half of trp was the weakest of the three. DNA sequences outside of these two regions must also play a role in promoter function. It is apparent that sequences beyond -39 of the trp promoter do not play an important role in expression, while changes in the transcribed, nontranslated region can alter gene expression. In vitro characterizations have suggested a general relationship between the rate of open complex formation and transition temperature among the promoters studied here. It was further shown that these parameters alone are not accurate predictors of in vivo promoter expression. Several of the promoters described here, trp-lac, trp, and trp-tet, will be very useful in studies requiring transcription or gene expression.