Changing the mechanism of transcriptional activation by phage lambda repressor

Abstract

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant, K(B)) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant, k(f)). lambda cI protein activates the PRM promoter by specifically increasing k(f). A positive control mutant, cI-pc2, is defective for activation because it fails to raise k(f). An Arg to His change in the sigma70 subunit of RNA polymerase was previously obtained as an allele-specific suppressor of cI-pc2. To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in sigma does not significantly alter K(B) or k(f) in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasing k(f). An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing K(B).

Figure 1

Sequence of the λ O_R region. The 5′ ends of RNAs made by natural transcription are shown, as well as trinucleotides made in abortive initiation reactions. Critical promoter elements are marked with asterisks. The O_R3-r2 mutation was used in assays in vitro.

Figure 2

System used to measure activation in vivo. Strains have only one form of σ⁷⁰, either wild-type or mutant, encoded by the rpoD gene at its normal locus in the E. coli chromosome (thick line). A prophage (thin line) at the λ attachment site produces a low level of cI protein encoded by various cI alleles fused to a weak, constitutive promoter. Effective combinations of σ and cI activate transcription of a lacZ reporter gene fused to wild-type λ P_RMO_R on a prophage at the P22 attachment site. λ cI protein does not control its own synthesis and does not control maintenance of lysogeny by either prophage.

Figure 3

Effect of wild-type cI and cI-pc2 on repression of P_R and activation of P_RM. The activities of P_R (A and C) and P_RM (B and D) in the presence of various concentrations of wild-type cI (•) or cI-pc2 (○) were measured by monitoring the rate of synthesis of abortive products by wild-type RNA polymerase holoenzyme (A and B) or holoenzyme containing σ⁷⁰-RH596 (C and D) as described in the text. The maximal rate for P_R (set at 100) corresponds to 361 or 337 CpApU per promoter per min in A and C, respectively; the nonactivated rate for P_RM (set at 1) corresponds to 71 or 81 UpApU per promoter per min in B and D, respectively.