Mode of DNA-protein interaction between the C-terminal domain of Escherichia coli RNA polymerase alpha subunit and T7D promoter UP element.
The C-terminal domain (CTD) downstream from residue 235 of Escherichia coli RNA polymerase alpha subunit is involved in recognition of the promoter UP element. Here we have demonstrated, by DNase I and hydroxyl radical mapping, the presence of two UP element subsites on the promoter D of phage T7, each located half and one-and-a-half helix turns, respectively, upstream from the promoter -35 element. This non-typical UP element retained its alphaCTD-binding capability when transferred into the genetic environment of the rrnBP1 basic promoter, leading to transcription stimulation as high as the typical rrnBP1 UP element. Chemical protease FeBABE conjugated to alphaCTD S309C efficiently attacked the T7D UP element but not the rrnBP1 UP element. After alanine scanning, most of the amino acid residues that were involved in rrnBP1 interaction were also found to be involved in T7D UP element recognition, but alanine substitution at three residues had the opposite effect on the transcription activation between rrnBP1 and T7D promoters. Mutation E286A stimulated T7D transcription but inhibited rrnBP1 RNA synthesis, while L290A and K304A stimulated transcription from rrnBP1 but not the T7D promoter. Taken together, we conclude that although the overall sets of amino acid residues responsible for interaction with the two UP elements overlap, the mode of alphaCTD interaction with T7D UP element is different from that with rrnBP1 UP element, involving different residues on helices III and IV.
Ozoline
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Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
Fujita
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N
Ishihama
A
A
D000596:Amino Acids / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014158:Transcription, Genetic
D000596:Amino Acids; D004269:DNA, Bacterial; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit