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pubmed:abstractText |
The abortive initiation assay [McClure, W. R. (1980) Proc. Natl. Acad. Sci. USA 77, 5634-5638] was used to study the effects of mutations on the activity of the P(RM) promoter of phage lambda in vitro. The transcription initiation properties of four mutant promoters were compared with those of wild-type P(RM) in the presence or absence of repressor (which activates P(RM)). Two kinetic parameters were measured: k(2), the rate constant for the transition between closed and open complexes, and K(B), the equilibrium constant for the initial binding of RNA polymerase to DNA (formation of closed complexes). The primary effect of repressor on wild-type initiation was stimulation of the isomerization reaction: k(2) increased about 7-fold. Both in the presence and in the absence of repressor, prmU31 and prmE104 (changes at nucleotides -33 and -38, respectively) reduced K(B) significantly without affecting k(2), indicating that these mutations affect polymerase binding but not the formation of open complexes. In contrast, prmE37 (a change at nucleotide -14) reduced k(2) significantly without affecting K(B). A fourth mutation, prmE93 (at nucleotide -39), is phenotypically Prm(-) primarily because it causes a defect in the O(B)2 operator site and, therefore, the mutant promoter is unable to respond normally to repressor. These results are consistent with the idea that the two regions of Escherichia coli promoters in which consensus sequences have been identified, the regions at nucleotides -35 and -10, may provide information for two discrete steps in transcription initiation.
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