Functional determinants of the Escherichia coli fis promoter: Roles of -35, -10, and transcription initiation regions in the response to stringent control and growth phase-dependent regulation

Escherichia coli Fis is a small DNA binding and bending protein that has be en implicated in a variety of biological processes. A minimal promoter sequ ence consisting of 43 bp is sufficient to generate its characteristic growt h phase-dependent expression pattern and is also subject to negative regula tion by stringent control. However, information about the precise identific ation of nucleotides contributing to basal promoter activity and its regula tion has been scant. In this work, 72 independent mutations were generated in the fis promoter (fis P) region from -108 to +78 using both random and s ite-directed PCR mutagenesis. beta-Galactosidase activities from mutant pro moters fused to the (trp-lac)W200 fusion on a plasmid were used to conclusi vely identify the sequences TTTCAT and TAATAT as the -35 and -10 regions, r espectively, which are optimally separated by 17 bp. We found that four con secutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the -35 region, but not in the -10 region, significantly reduc ed the response to stringent control. Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G ) profoundly altered expression such that high levels of fis P mRNA were de tected during late logarithmic and early stationary phases. A less dramatic effect was seen with improvements in the -10 and -35 consensus sequences. These results suggest that the acute growth phase-dependent regulation patt ern observed with this promoter requires an inefficient transcription initi ation process that is achieved with promoter sequences deviating from the - 10 and -35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP .