RNA Polymerase a and s Subunits Participate in Transcription of the Escherichia coli uhpT Promoter

Fundamental questions in bacterial gene regulation concern how multiple regulatory proteins interact with the transcription apparatus at a single promoter and what are the roles of protein contacts with RNA polymerase and changes in DNA conformation. Transcription of the Escherichia coli uhpT gene, encoding the inducible sugar phosphate transporter, is dependent on the response regulator UhpA and is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to multiple sites in the uhpT promoter between positions 280 and 232 upstream of the transcription start site, and CAP binds to a single site centered at position 2103.5. The role in uhpT transcription of portions of RNA polymerase Es holoenzyme which affect regulation at other promoters was examined by using series of alanine substitutions throughout the C-terminal domains of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613). Alanine substitutions that affected in vivo expression of a uhpT-lacZ transcriptional fusion were tested for their effect on in vitro transcription activity by using reconstituted holoenzymes. Consistent with the binding of UhpA near the 235 region, residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient uhpT expression in response to UhpA alone. Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (aCTD) were important for uhpT transcription even in the presence of CAP. Substitutions at several other positions had effects in cells but not during in vitro transcription with saturating levels of the transcription factors. Two DNase-hypersensitive sites near the upstream end of the UhpA-binding region were seen in the presence of all three transcription factors. Their appearance required functional aCTD but not the presence of upstream DNA. These results suggest that both transcription activators depend on or interact with different subunits of RNA polymerase, although their role in formation of proper DNA geometry may also be crucial.