Regulation of transcription in bacteriophage T5-infected Escherichia coli

Szabó, C. Ákos; Dharmgrongartama, B.; Moyer, Richard W.

doi:10.1021/bi00676a018

Cited by 28 publications

(26 citation statements)

References 41 publications

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“…This complex becomes successively modified during pre-early phase by gene products A3 (56) and Al (40) to allow transcription of early genes and during early phase by gene product C2 and a 15K protein (57) to allow transcription of late genes. Transcription of late genes, in addition, requires the activity of host DNA gyrase (8) and T5 gene products D5 and D15 (41).…”

Section: Resultsmentioning

confidence: 99%

Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein

Krauel

Heller

1991

J Bacteriol

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Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5. In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene. In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome. This analysis also showed that oad has little or no homology with hrs, the analogous BF23 gene. We were able to overproduce a protein that comigrates with pb5 after fusing a 2-kb segment containing oad to a phage T7

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Section: Resultsmentioning

confidence: 99%

Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein

Krauel

Heller

1991

J Bacteriol

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“…This transcription is known to require the products of both the T5 "early" genes, C2 and D15 (3,4). Szabo et al (19) have provided some evidence supporting the proposal of Chinnadurai and McCorquodale (4) that the C2 gene product is a phage "sigma-like" factor, which, as a consequence of binding to Escherichia coli RNA polymerase, permits the host enzyme to recognize the late class of T5 DNA promoters. The gene D15 product is known to be a nuclease (6,9).…”

mentioning

confidence: 81%

“…Single amber mutants of T5+ in either gene C2 (T5C2amM142a) or gene D15 (T5D15amH50) were provided by D. J. Mc-Corquodale. The protein products produced by phage genes D15 and C2 are thought to be a nuclease (6,9) and a "a" factor, which interacts with E. coli RNA polymerase (4,19), respectively.…”

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confidence: 99%

“…Sample preparation and agarose gel analysis of radiolabeled T5 DNA synthesized in infected cells. Bacteria were grown in T-MGM medium and synchronously infected with T5+ by the procedure of Szabo et al (19) with the following modifications. After growth and collection of the cells by centrifugation, the cellular pellet was suspended, before infection, in T-MGM buffer lacking thymine to increase the amount of [3H]thymidine incorporated into replicating T5 DNA.…”

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confidence: 99%

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Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA

Moyer¹,

Rothe²

1977

J Virol

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The processing of newly replicated concatameric T5 DNA into both single stranded DNA chains of unit length and single-stranded fragments of sizes comparable to those found in mature T5 virion DNA occurs in the absence of late T5 protein synthesis. The formation of unit-length, single-stranded DNA chains does not require the early T5 gene D15 nuclease; however, the subsequent formation of the single-stranded fragments does require that the D15 nuclease be functional. A reexamination of the properties of the purified D15 nuclease under a variety of conditions showed that, in addition to functioning as a 5'->3' exonuclease, the enzyme can also introduce endonucleolytic scissions into mature T5 DNA in a reaction that requires duplex T5 DNA and preexisting, singlestranded interruptions.The location, origin, and function of the specific, single-stranded interruptions (nicks) contained within the genome of bacteriophage T5 DNA have attracted considerable attention for a number of years. The proposed function of the nicks currently centers around their putative role in transcription of late T5 genes. This transcription is known to require the products of both the T5 "early" genes, C2 and D15 (3, 4). Szabo et al. (19) have provided some evidence supporting the proposal of Chinnadurai and McCorquodale (4) that the C2 gene product is a phage "sigma-like" factor, which, as a consequence of binding to Escherichia coli RNA polymerase, permits the host enzyme to recognize the late class of T5 DNA promoters. The gene D15 product is known to be a nuclease (6,9). Enzymatic studies of partially purified D15 nuclease have shown that the protein possesses 5'-*3' exonuclease activity on both singleand double-stranded DNA (6,14). T5 strains containing amber mutations in gene D15 not only fail to initiate late transcription, but also fail to introduce the single-stranded interruptions into newly replicated phage DNA (7).There are at least two mechanisms by which the gene D15 product could control the introduction of nicks into replicating T5 DNA. The first, and the most direct, hypothesis would be that the nuclease encoded by gene D15 can also function as a very specific endonuclease and thereby introduce the nicks into replicating DNA. This hypothesis would also be consistent with the proposal that a nicked DNA template is required for late transcription (11). The enzyme must, however, possess very little, if any, gen-eral endonucleolytic activity because the protein is unable to cleave either doubleor singlestranded circular OX174 DNA (6). Alternatively, the introduction of nicks into T5 DNA might be under more indirect control of the D15 gene. For example, gene D15 amber mutants are unable to synthesize late T5 RNA and proteins, and nicking would then be prevented with D15 mutants if either a late protein or complex of late proteins were required for this process. Our results favor the first hypothesis, and we present evidence suggesting that the D15 nuclease plays a direct role in introducing nicks into replicating T5 DNA. Nicking ...

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“…Late mRNA transcripts,on the contrary, only begin to appear some 10 to 12 minutes after infection. Experiments using rifampicin (4) and RNA polymerase purified from T5 infected cells (5,6) suggest that the E.coli RNA polymerase serves to transcribe the T5 DNA throughout infection though,at late times,it does so in association with T5 coded polypeptides.Analysis of T5 mutants shows that at least three T5 gene products (C2, D5, D15) are needed for late transcription.The C2 gene codes for a 90K polyneptide that binds to RNA polymerase, as do two unidentified polypeptides of 11K and 15K (5,6). The D15 product is a 5'-3' exonuclease (7,8) able to introduce endonucleolytic scissions in T5 DNA (9).…”

Section: Introductionmentioning

confidence: 99%