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Dissection of the bacteriophage T4 late promoter complex.

ABSTRACT: Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator is gp45, the viral replisome's sliding clamp; two sliding-clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) sigma subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding-clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ: only the gp33-gp45 linkage is essential for activation, while loss of the gp55-gp45 linkage impairs but does not abolish activation. Formation of transcription-ready promoter complexes by the sliding-clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret the relationship of these findings and our prior analysis to the common model of transcriptional initiation in bacteria in terms of two parallel pathways, with two RNAP holoenzymes and two DNA templates: (1) gp55-RNAP and the T4 late promoter execute basal transcription; (2) gp55-gp33-RNAP and the T4 late promoter with its mobile enhancer, the T4 sliding clamp, execute activated transcription. gp55 and gp33 perform sigma-like functions, gp55 in promoter recognition and gp33 (as well as gp55) in enhancer recognition. gp33 operates the switch between these two pathways by repressing basal transcription.

SUBMITTER: Nechaev S

PROVIDER: S-EPMC2536758 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Dissection of the bacteriophage T4 late promoter complex.

Nechaev Sergei S Geiduschek E Peter EP

Journal of molecular biology 20080407 3

Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator is gp45, the viral replisome's sliding clamp; two sliding-clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) sigma subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The ...[more]

PMID: 18455735

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Project description:Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the "cell-puncturing device," combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages-the most abundant and among the most ancient biological entities on Earth.

Dataset Information

Dissection of the bacteriophage T4 late promoter complex.

Publications

Dissection of the bacteriophage T4 late promoter complex.

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