Direct and indirect effects of mutations in lambda PRM on open complex formation at the divergent PR promoter.
A detailed kinetic analysis demonstrates that, in vitro, mutations in the PRM promoter of bacteriophage lambda can increase the rate of open complex formation at the divergent, lytic promoter PR in either of two ways. (1) PRM- mutations, typified by PRMKM11, indirectly stimulate PR by eliminating interference from RNA polymerase (RNAP) molecules bound at wild-type PRM. This effect can be observed only when PR is itself mutated because open complexes normally form so rapidly at wild-type PR that they are unaffected by PRM. It has been shown previously that PR and PRM can be occupied simultaneously by RNAP, suggesting that interference from PRM is mediated at a step subsequent to binding of RNAP to PR. This conclusion is supported by kinetic data, which indicate that inactivating PRM affects PRx3 by increasing kf, the rate of isomerization of closed to open complexes, four- to fivefold. (2) In addition to its indirect effect, the mutation PRM116, which is located at -33 with respect to PRM and -50 with respect to PR, directly increases the intrinsic strength of PR. PRM116 increases from 11 to 12 the number of A:T or T:A base-pairs in a 12 bp AT-rich sequence located between 47 and 58 bp upstream from PR; we suggest that this upstream sequence contributes directly to PR promoter strength. We also show that the PRx3 mutation causes a 100-fold decrease in kf. This result indicates that the -35 consensus region plays a major role in the isomerization of closed to open complexes at PR.
Department of Biological Sciences, University of Iowa, Iowa City 52242.
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Fong
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Woody
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Gussin
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic
D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases