Direct and Indirect Effects of Mutations in λ PRM on Open Complex Formation at the Divergent PR Promoter

Abstract

A detailed kinetic analysis demonstrates that, in vitro, mutations in the P RM promoter of bacteriophage λ can increase the rate of open complex formation at the divergent, lytic promoter P R in either of two ways. (1) P - RM mutations, typified by P RM KM11, indirectly stimulate P R by eliminating interference from RNA polymerase (RNAP) molecules bound at wild-type P RM. This effect can be observed only when P R is itself mutated because open complexes normally form so rapidly at wild-type P R that they are unaffected by P RM . It has been shown previously that P R and P RM can be occupied simultaneously by RNAP, suggesting that interference from P RM is medicated at a step subsequent to binding of RNAP to P R. This conclusion is supported by kinetic data, which indicate that inactivating P RM affects P R x3 by increasing k f, the rate of isomerization of closed to open complexes, four- to fivefold. (2) In addition to its indirect effect, the mutation P RM116, which is located at -33 with respect to P RM and -50 with respect to P R , directly increases the intrinsic strength of P R . P RM116 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 P R; we suggest that this upstream sequence contributes directly to P R promoter strength. We also show that the P R x3 mutation causes a 100-fold decrease in k f. This result indicates that the -35 consensus region plays a major role in the isomerization of closed to open complexes at P R.