AMINO-ACID-RESIDUES IN THE ALPHA-SUBUNIT C-TERMINAL DOMAIN OF ESCHERICHIA-COLI RNA-POLYMERASE INVOLVED IN ACTIVATION OF TRANSCRIPTION FROM THE MTR PROMOTER

To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the alpha-subunit of RNA polymer ase in TyrR-mediated activation of the mtr promoter, we have carried o ut in vitro transcription experiments using a set of mutant RNA polyme rases with a supercoiled mtr template, Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K2 97A suggested the presence of a possible UP element associated with th e mtr promoter, Mutational studies have revealed that an AT-rich seque nce centered at -41 of the mtr promoter (SeqA) functions like an UP el ement, In vivo and in vitro analyses using a mutant mtr promoter carry ing a disrupted putative UP element showed that this AT-rich sequence is responsible for interactions with the alpha-subunit which influence transcription in the absence of TyrR protein, However, the putative U P element is not needed for activator-dependent activation of the mtr promoter by TyrR and phenylalanine. The results from in vitro studies indicated that the alpha-subunit residues leucine-262, arginine-265, a nd lysine-297 are critical for interaction with the putative UP elemen t of the mtr promoter and play major roles in TyrR-dependent transcrip tion activation, The residues at positions 258, 260, 261, 268, and 270 also play important roles in TyrR-dependent activation, Other residue s, at positions 259, 263, 264, 266, 269, 271, 273, 275, and 298, appea r to play less significant roles or no role in activation of mtr trans cription.