During
infection of Escherichia coli, the phage T4 early
protein AsiA inhibits open complex formation by the
RNA polymerase holoenzyme Efinal
sigma(70)
at -10/-35 bacterial promoters through binding to region 4.2 of the final
sigma(70) subunit. We used the -10/-35 lacUV5 promoter to study the properties of the Efinal
sigma(70). AsiA complex in the presence of the
glutamate anion. Under these experimental conditions, inhibition by AsiA was significantly decreased. KMnO(4) probing showed that the observed residual transcriptional activity was due to the slow transformation of the ternary complex Efinal
sigma(70). AsiA.lacUV5 into an open complex. In agreement with this observation, affinity of the
enzyme for the promoter was 10-fold lower in the ternary complex than in the binary complex Efinal
sigma(70).lacUV5. A tau plot analysis of abortive transcription reactions showed that AsiA binding to Efinal
sigma(70) resulted in a 120-fold decrease in the second-order on-rate constant of the reaction of Efinal
sigma(70) with lacUV5 and a 55-fold decrease in the rate constant of the isomerization step leading to the open complex. This ternary complex still responded to activation by the cAMP.
catabolite activator protein complex. We show that compensatory Efinal
sigma(70)/promoter upstream contacts involving the C-terminal domains of alpha subunits in Efinal
sigma(70) become essential for the binding of Efinal
sigma(70). AsiA to the lacUV5 promoter.