Regulation of Gene Action in the Development of Lytic Bacteriophages

Rabussay, Dietmar; Geiduschek, E. Peter

doi:10.1007/978-1-4684-2715-8_1

Cited by 24 publications

(23 citation statements)

References 776 publications

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“…Whether translation of late T5 genes also depends on T5-coded proteins is not known. However, translation of late genes of bacteriophage T4 has also been thought to depend on T4-coded factors (16).…”

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confidence: 99%

Cloning and expression of the ltf gene of bacteriophage T5

Heller¹,

Krauel²

1986

J Bacteriol

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The unique 5-kilobase BamHI fragment of bacteriophage T5 was cloned into plasmid pBR322. Location of the intact ltfgene on the cloned fragment was demohstrated by (i) complementation of the Itfmutation of phage T5hd-2, (ii) identification of a plasmid-coded polypeptide of the same molecular weight as the polypeptide forming the L-shaped tail fibers, which binds to anti-T5 antibodies; and (iii) analyses of transposon TnlOOO insertions.

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confidence: 99%

Cloning and expression of the ltf gene of bacteriophage T5

Heller¹,

Krauel²

1986

J Bacteriol

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“…Some classes of middle and late phage genes can be transcribed in the absence of ai (28,29). It should be possible to confirm that coliphage T4 late genes are expressed without a (29) by using strain 285c and shifting to nonpermissive temperature after T4 early genes have been expressed.…”

Section: Discussionmentioning

confidence: 99%

Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

Harris¹,

Heilig²,

Martinez³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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A gene affecting the or subunit of DNA-dependent RNA polymerase is tightly linked to dnaG at 66 min on the Escherichia coli chromosome. In order to create an easily selectable marker in this region, we inserted transposon-10, which carries a gene determining resistance to tetracycline (tet) near 66 minm and the order toC-dnaG--tet was determined. We used frequency of cotransduction with tet as a criterion to screen a collection of spontaneous temperature-sensitive Escherichia coli mutants that might affect the a subunit. One such mutant was found to map at the or locus. The a subunit isolated from this mutant is unstable at 460C in vitro and has an altered electrophoretic mobility. The temperature sensitivity of RNA synthesis in this mutant indicates that most transcription in E. coli is cr dependent.The a subunit of Escherichia coli DNA-dependent RNA polymerase is responsible for much of the specificity of in vitro transcription (1, 2). For example, RNA polymerase core enzyme (lacking a subunit) binds weakly to many sites on T7 phage DNA in vitro, whereas RNA polymerase holoenzyme (containing a subunit) binds strongly and only to those sites at which in vivo transcription begins (3).Elucidation of the role of the a protein in the specificity of gene expression has been delayed, however, by the lack of a subunit mutants. Previous genetic studies on the electrophoretic differences among the a proteins of enteric bacteria suggested that the structural gene for a maps at about 66 min on the E. coli genome (4, 5). In order to rapidly screen potential a mutants in this region of the genome, we have inserted the translocatable tetracycline-resistance (tet) element TnlO near the a gene. By using this readily selectable marker for rapid mapping of mutants, we have identified a spontaneous temperature-sensitive (ts) mutant that produces an altered a protein and is deficient in the synthesis of RNA at nonpermissive temperatures.MATERIALS AND METHODS Media, Buffers, and Chromatographic Materials. LB broth (6) supplemented with 0.4% glucose was used; the NaCl concentration was adjusted to 0.1 M. L plates consist of LB broth and 2% Difco agar. LC plates contain, in addition, 5 mM CaCd2 but 1.5% agar. Davis minimal medium is described by Calendar and Lindahl (7). Buffer P50 is described by Gonzalez et al. (8) and TGED buffer is described by Burgess and Jendrisak (9). TPG-CAA medium (10) enriched with 0.8% glucose, 0.01% arginine, 0.5 mM CaCI2, and 20 amino acids and trace metals each at 1 ttg/ml was used for radioactive labeling. Bio-Gel A-1.5m used for gel filtration and the ion-exchange material Bio-Rex 70 were purchased from Bio-Rad. (11), selecting for tetracycline-resistance and the ability to grow at 42'C (dnaG +). This procedure yielded transductants with a tet gene that is highly cotransducible with dnaG. C-2359 is one of these.Purification of RNA Polymerase. RNA polymerase holoenzyme was purified according to the procedure of Burgess and Jendrisak (9). After the enzyme was eluted from the A-1.5m column, i...

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“…It has been hypothesized that these phages make their own RNA polymerases to become independent of their hosts for transcription (Rabussay and Geiduschek 1977). N4 could be seen as an extreme example of this strategy because injection of the virion-associated RNA polymerase bypasses the need for the host transcriptional machinery early in infection.…”

Section: Ssb Interactions With Supercoiled Template At the Virion Rnamentioning

confidence: 99%

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Markiewicz¹,

Malone²,

Chase³

et al. 1992

Genes Dev.

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Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coil single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.[Key Words: Coliphage N4; RNA polymerase; single-stranded DNA-binding protein; transcriptional activation; template supercoiling] Bacteriophage N4 is a double-stranded DNA virus specific for Escherichia coli K 12 strains. Transcription of its genome is regulated through the sequential activity of three distinct RNA polymerases (for review, see Kiino and Rothman-Denes 1988). Early N4 transcripts are synthesized by a rifampicin-resistant, N4-coded RNA polymerase, which is carried within the virion and injected into the cell at the start of infection (Falco et al. 1977). N4 middle transcripts are synthesized by a second N4-coded RNA polymerase (N4 RNA polymerase II) (Zehring and Rothman-Denes 1983; . Late in infection, the N4-coded, single-stranded DNA-binding protein directs the host RNA polymerase to transcribe the N4 structural genes (Zivin et al. 1981; N. Baek, M. Choi and L. Rothman-Denes, in prep.) Purified virion RNA polymerase shows peculiar template specificity. It is unable to use native duplex genomic N4 DNA as a template (Falco et al. 1978(Falco et al. , 1980 polymerases, it transcribes denatured or single-stranded, promoter-containing templates accurately and efficiently (Haynes and Rothman-Denes 1985; Glucksmann et al. 1992). This result suggests that the structure of N4 DNA must be altered upon entry into the cell to allow transcription by the virion RNA polymerase. It is likely that the N4 virion RNA polymerase utilizes a supercoiled template because active host DNA gyrase is required for N4 early RNA synthesis (Falco et al. 1978). However, in vitro, supercoiled, promoter-containing templates do not support virion RNA polymeras...

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Regulation of Gene Action in the Development of Lytic Bacteriophages

Cited by 24 publications

References 776 publications

Cloning and expression of the ltf gene of bacteriophage T5

Cloning and expression of the ltf gene of bacteriophage T5

Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

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