T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis

The consensus sequence of 14 early promoters differs in length, sequence an d degree of conservation from that of Escherichia coil sigma (70) promoters . The enzyme interacting with these promoters, and transcribing the T4 geno me, is native host RNA polymerase, which is increasingly modified by the ph age-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very a ctive process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and t he Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of wh ich promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coil promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensu s. Second, mutations were introduced into the highly conserved regions of t he T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plas mid pKWIII and vector pTKRI, which expresses Alt in E. coil, constitutes a test system that allows comparison of the transcriptional activities of pha ge and bacterial promoters, in the presence of native, or alternatively ADP -ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RN A polymerase. The -10, -16, -42 and -52 regions are important for transcrip t initiation with the native polymerase. To facilitate acceleration of tran scription, the ADP-ribosylated enzyme requires not only the integrity of th e -10, -16 and -35 regions, but also that of position -33, and even more im portantly, maintenance of the upstream promoter element at position -42. Th e latter positions introduced into the E. coil Ptac promoter render this mu tant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 ear ly promoters.