Characterization of Bacteriophage T4 Early Promotersin Vivowith a New Promoter Probe Vector

K, Wilkens; Rüger, Wolfgang

doi:10.1006/plas.1996.0013

Cited by 19 publications

(12 citation statements)

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“…Significantly, primer extension analysis of the nrdA-a early promoter showed that the promoter remained active over the course of the phage infection and that the initiating nucleotide for transcription was C-22. Both of these observations are in contrast to T4 early promoters, which usually initiate at an A nucleotide (32,61) and which are active 1 to 3 min post-T4 infection (35,40). Likewise, the late promoter upstream of Aeh1 mobE is active 15 min postinfection, a significant delay compared with T4 late promoters, which are active 7 min postinfection (35,40).…”

Section: Discussionmentioning

confidence: 93%

Multiple Controls Regulate the Expression of mobE , an HNH Homing Endonuclease Gene Embedded within a Ribonucleotide Reductase Gene of Phage Aeh1

Gibb

Edgell

2007

J Bacteriol

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Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon (nrd) that has been invaded by an HNH family homing endonuclease, mobE. In Aeromonas hydrophila phage Aeh1, mobE has inserted into the large-subunit gene (nrdA) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b. This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aeh1 mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aeh1-specific late promoter drives expression of mobE, but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.Homing endonucleases are a distinctive class of site-specific yet sequence-tolerant DNA endonucleases that promote the mobility of themselves and surrounding genomic sequence to genomes lacking the endonuclease by a process termed homing (reviewed in reference 3). Homing endonuclease genes are often found within self-splicing group I or II introns (3, 30) or inteins (20, 21), but many bacterial and phage genomes encode a large number of so-called freestanding endonucleases, i.e., the genomes carry endonuclease genes that are not obviously encoded within self-splicing elements (13,29,40,54). Experimental evidence to date suggests that freestanding endonucleases are also mobile genetic elements, promoting their spread to genomes lacking the endonuclease by a double-strand break (DSB) repair pathway termed intronless homing (4, 28, 33, 51). Database surveys of sequenced bacterial and phage genomes have revealed that freestanding endonucleases are more abundant than intron-or intein-encoded versions, which is particularly evident in the T-even-like phages (29,40,44,54). Phage T4, for instance, is infested with 15 homing endonucleases, representing ϳ10% of the coding potential of the genome; 13 of these endonucleases are freestanding (40).Whereas the mobility pathways of intron-encoded and freestanding endonucleases are well described (4,33,41,46), comparatively little is known regarding the regulation of homing ...

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Section: Discussionmentioning

confidence: 93%

Multiple Controls Regulate the Expression of mobE , an HNH Homing Endonuclease Gene Embedded within a Ribonucleotide Reductase Gene of Phage Aeh1

Gibb

Edgell

2007

J Bacteriol

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“…Transcription start sites of many of the early promoters have been mapped by primer extension off of mRNA from T4-infected cells and/or from promoter-cloning vectors (reviewed in reference 1169). The 39 characterized Pe sequences (1168,1169) are noted in Table 1 and have been analyzed using the information content software developed by Schneider and Stephens (966). The sequence logos, maximizing the alignment at the Ϫ10 region and, independently, at the Ϫ35 region, are shown in Fig.…”

Section: Early Transcriptionmentioning

confidence: 99%

“…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).…”

Section: Early Transcriptionmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

688

801

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SUMMARY 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.

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“…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 .…”

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confidence: 99%

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confidence: 99%

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

Tiemann

Depping

Gineikiene³

et al. 2004

J Bacteriol

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Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Altand ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.Posttranslational ADP-ribosylation of proteins is catalyzed by ADP-ribosyltransferases (ADP-RTs), which have been identified in viral, bacterial, and eukaryotic systems. Transfer of the ADP-ribose moiety from the substrate NAD ϩ to a specific amino acid residue, frequently histidine or arginine within a target protein, modulates the activity of the acceptor. ADP-ribosylation changes the electrostatic potential of a target protein by introducing two phosphate groups and may affect protein-DNA as well as protein-protein interactions. ADP-RTs were initially discovered as the exotoxins of pathogenic bacteria. Therefore, most of our knowledge concerning these proteins has been gained by biochemical, genetic, and structural studies performed on bacterial toxins, such as those produced by Corynebacterium diphtheriae (15, 30), Bordetella pertussis (34), Vibrio cholerae (46), Pseudomonas aeruginosa (33), and Escherichia coli (45). New putative bacterial toxins were found to be encoded in the genomes of Streptococcus pyogenes and Salmonella enterica serovar Typhi (50).To date, the family of ADP-RTs comprises more than 40 enzymes, including bacterial exotoxins as well as a variety of other enzymes, such as the eukaryotic mono-and poly-ADPRTs, which include T-cell differentiation alloantigens like RT6 (9), poly-ADP-RTs (56), the dinitrogenase reductase regulation factor DraT (51,77), and enzymes encoded by T-even bacteriophages. The bacteriophage T4 gene products Alt (76 kDa), ModA (23 kDa), and ModB (24 kDa) appear to actively regulate gene expression during the transition from host t...

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Characterization of Bacteriophage T4 Early Promotersin Vivowith a New Promoter Probe Vector

Cited by 19 publications

References 0 publications

Multiple Controls Regulate the Expression of mobE , an HNH Homing Endonuclease Gene Embedded within a Ribonucleotide Reductase Gene of Phage Aeh1

Multiple Controls Regulate the Expression of mobE , an HNH Homing Endonuclease Gene Embedded within a Ribonucleotide Reductase Gene of Phage Aeh1

Bacteriophage T4 Genome

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

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