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

Abstract

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli σ 70 promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage 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 RNA polymerase. The N10, N16, N42 and N52 regions are important for transcript initiation with the native polymerase.To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the N10, N16 and N35 regions, but also that of position N33, and even more importantly, maintenance of the upstream promoter element at position N42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADPribosylated RNA polymerase, like T4 early promoters.

ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

“…4, the Alt-modified E. coli RNA polymerase incorporated about twice as much radioactivity into RNA with T4 DNA as the unmodified enzyme incorporated. This observation confirmed previous experiments which provided evidence that the Alt-catalyzed ADP-ribosylation of RNA polymerase enhances transcription from T4 early pro- moters (60,73). On the other hand, the ModA-modified polymerase incorporated only 72% of the radioactivity that was found in the test with the unmodified enzyme and T4 DNA, underlining the negative influence of the ModA-catalyzed modification on the transcription from T4 early DNA.…”

“…Isolation and partial characterization of a recombinant Alt protein (GenBank accession number X15811), now available in larger amounts, revealed that host RNA polymerase subunits ␤, ␤Ј, and are also ADP-ribosylated, as are a number of other E. coli proteins (36, 37). ADPribosylation of RNA polymerase, as catalyzed by Alt, triggers the preferred transcription from T4 early promoters as possibly the first step by which T4 gains control over the metabolism of the host cell (60,72,73).…”

“…Isolation and partial characterization of a recombinant Alt protein (GenBank accession number X15811), now available in larger amounts, revealed that host RNA polymerase subunits ␤, ␤Ј, and are also ADP-ribosylated, as are a number of other E. coli proteins (36, 37). ADPribosylation of RNA polymerase, as catalyzed by Alt, triggers the preferred transcription from T4 early promoters as possibly the first step by which T4 gains control over the metabolism of the host cell (60,72,73).The ModA and ModB proteins are encoded by two neighboring T4 genes (44, 63) under control of the strong early promoter Pe 12.8, and their activities are directed against different cellular target proteins. As first demonstrated by Skorko et al (57), ModA-catalyzed ADP-ribosylation, in contrast to Alt activity, targets both ␣ subunits of RNA polymerase at residue Arg 265 .…”

Bacteriophage T4 Genome

“…Mutation analyses reveal that T4 early promoters interact strongly with unmodified RNAP and even better, in most cases, with RNAP in which only one of the ␣ subunits is ADP-ribosylated. In particular, base position Ϫ33 of the T4 promoter and the A-rich UP element at position Ϫ42 contribute to the strong interactions with ADP-ribosylated RNAP of T4-infected cells (1026). Therefore, Alt presumably contributes to the preferential transcription from T4 promoters after infection (1168,1170).…”

High-Throughput Screening of RNA Polymerase Inhibitors Using a Fluorescent UTP Analog

“…T4 phage DNA is rich in adenosine and thymidine nucleotides (64% AT) with 40 strong early promoters. 10 The consensus sequence at the transcription start site has an "A" followed by a pyrimidine residue, which may favor abortive transcription, leading to a significant increase in fluorescence as a result of incorporation of UMP from (γ-AmNS) UTP. This was probably reflected in the maximum inhibition by rifampicin not reaching more than 70% to 80% in this fluorescent assay.…”