Temporal Regulation of Gene Expression of the Escherichia coli Bacteriophage phiEco32 (original) (raw)
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MGG Molecular & General Genetics, 1983
Fractionation of pulse-labeled RNA extracted from E. coli cells infected with phage fl and hybridization of this RNA to fl DNA reveals that very large species are synthesized on the phage genome. Hybridization of the RNA to specific fragments of fl DNA shows that, in the infected cell, at least one mRNA is present into which the sequences of genes III, VI, and I are all transcribed together. This result fully explains the polar effect shown by gene III mutants on the expression of genes VI and I .
Journal of Biological Chemistry, 1997
The AsiA protein of bacteriophage T4 binds to the 70 subunit of Escherichia coli RNA polymerase and plays a dual regulatory role during T4 development: (i) inhibition of host and phage early transcription, and (ii) coactivation of phage middle-mode transcription, which also requires the T4 DNA binding transcriptional activator, MotA. We report that the interaction between AsiA and 70 occurs with a 1:1 stoichiometry. When preincubated with RNA polymerase, AsiA is a potent inhibitor of open complex formation at the lac UV5 promoter, whereas it does not perturb preformed open or intermediate promoter complexes. DNase I footprinting and electrophoretic mobility shift analyses of RNA polymerase-DNA complexes formed at the T4 early promoter P15.0 show that AsiA blocks the initial RNA polymerase binding step that leads to the formation of specific closed promoter complexes. A contrasting result is obtained on the T4 middle promoter PrIIB2, where AsiA stimulates the formation of both closed complexes and open complexes. Therefore, we propose that AsiA modulates initial DNA binding by the RNA polymerase, switching promoter usage at the level of closed complex formation.
Nucleic acids research, 2017
Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.
Translational control of the expression of bacteriophage T7 gene 0.3
Journal of Molecular Biology, 1978
When Escherichia coli are infected at 43°C with a bacteriophage T7 mutant that produces a temperature-sensitive RNA polymerase (ts342), the rate of transcription of the T7 late genes is reduced three-to fourfold below the rate of transcription in cells infected with wild-type T7. The reduction in T7 late mRNA concentration in cells infected with ts342 is accompanied by the overproduction at late times of at least one T7 early protein, the gene O-3 protein. Despite the difference in T7 late mRNA concentration in cells infected with wild-type T7 and ts342 at 43"C, T7 late proteins are synthesized at the same rate in the two infectedcell cultures. These findings support an hypothesis of discrimination against 0.3 mRNA translation in favor of translating T7 late messages when mRNA is in excess of the protein synthetic machinery of the cell.
Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein
Journal of Molecular Biology, 2011
E. coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). Gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the −10 promoter element. The inhibition of RNAP interactions with singlestranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.
The EMBO Journal, 1996
Phage 029 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Argl20 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Argl20, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Argl20 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription.
Identification of Upstream Sequences Essential for Activation of a Bacteriophage P2 Late Promoter
Journal of Bacteriology, 2003
We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P F , which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 Delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for Delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the ؊35 region of the promoter. Introduction of a 70 ؊35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by Delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor 70 contacts and helps to recruit RNA polymerase holoenzyme.
The Role of the T7 Gp2 Inhibitor of Host RNA Polymerase in Phage Development
Journal of Molecular Biology, 2010
Bacteriophage T7 relies on its own RNA polymerase (RNAp) to transcribe its middle and late genes. Early genes, which include the viral RNAp gene, are transcribed by the host RNAp from three closely spaced strong promoters --A1, A2, and A3. One middle T7 gene product, gp2, is a strong inhibitor of the host RNAp. Gp2 is essential and is required late in infection, during phage DNA packaging. Here, we explore the role of gp2 in controlling host RNAp transcription during T7 infection. We demonstrate that in the absence of gp2 early viral transcripts continue to accumulate throughout the infection. Decreasing transcription from early promoter A3 is sufficient to make gp2 dispensable for phage infection. Gp2 also becomes dispensable when an antiterminating element boxA, located downstream of early promoters, is deleted. The results thus suggest that antiterminated transcription by host RNAp from the A3 promoter is interfering with phage development and that the only essential role for gp2 is to prevent this transcription.