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Abstract
The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.
Highlights
FIS is a 98-amino-acid DNA-binding protein that activates transcription from Escherichia coli rRNA promoters [30], several tRNA promoters [23, 24], and the proP promoter [35]
The region between the FIS sites and the Ϫ35 hexamer constitutes a third promoter recognition element that interacts with the ␣ subunit of RNA polymerase (RNAP) and stimulates transcription another 30-fold [28, 29]. rrn P1 promoters are regulated in response to nutritional conditions by at least two distinct systems, growth rate-dependent control and stringent control
In strains lacking active FIS, rrn P1 core promoter activity increases to compensate for the decrease in transcription activation of the seven rRNA operons [30]. This derepression is a consequence of a feedback mechanism that regulates the rate of rRNA and tRNA synthesis in the cell in response to the levels of translationally competent ribosomes [3, 11, 13, 36]
Summary
FIS is a 98-amino-acid DNA-binding protein that activates transcription from Escherichia coli rRNA promoters [30], several tRNA promoters [23, 24], and the proP promoter [35]. We identified a class of mutant proteins unable to stimulate rrnB P1 transcription yet able to bind and bend DNA in vitro This and other information [2, 22] indicated that an additional FIS function, presumably interaction with RNAP, is likely to be required for transcription activation. Characterization of the mutant protein with a G-to-S mutation at position 72 (G72S) in vitro confirmed that this mutant protein is defective in transcription activation, yet its DNA binding and bending characteristics are unaffected This suggests that the loop between ␣-helices B and C of FIS [37] may define a region responsible for interactions with RNAP
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