Controls and polarity of transcription during bacteriophage T4 development

Guha, Anirban; Szybalski, Waclaw; Salser, Winston; Bolle, A.; Geiduschek, E. Peter; Pulitzer, John F.

doi:10.1016/0022-2836(71)90054-4

Cited by 85 publications

(46 citation statements)

References 34 publications

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“…This is not inconsistent with the significant amount of gp55-independent transcription which normally occurs late in infection (17) and which is independent of phage replication (45). It is also consistent with the compositional complexity of RNA polymerase and RNA polymerase-binding proteins in T4 development (42).…”

Section: Discussionsupporting

confidence: 76%

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

1988

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Concurrent viral replication is normally required to activate bacteriophage T4 late promoters; replication is thought to provide a template structure which is competent for late transcription. Transcription from plasmid-borne T4 late promoters, however, is independent of replication in vivo and in vitro. In this work, we have shown that, when the late gene 23 promoter is located on a plasmid, its utilization in vivo depends upon the ability of host DNA gyrase to maintain some degree of negative superhelicity. This suggests that an alternative pathway exists for activation of late promoters: DNA which is under sufficient negative torsional stress is already competent for late transcription. We also describe a method for isolating ternary complexes of plasmid DNA, RNA polymerase, and nascent RNA which have initiated transcription in vivo. The topoisomer distribution of such ternary complexes prepared from T4-infected cells showed that, late in infection, transcriptional activity resides primarily in the subset of the plasmid population with the most negatively supercoiled topoisomers. However, the overall transcriptional pattern in these ternary complexes indicated that both vector and T4 sequences are actively transcribed. Much of this transcriptional activity could be independent of gp55, the T4-specific RNA polymerase-binding protein that confers late promoter recognition.During infection of Escherichia coli by bacteriophage T4, a program of modifications to the host RNA polymerase generates three major temporal classes of phage transcripts (reviewed in references 7, 15, and 42). Late transcription requires the products of T4 genes 33, 45, and 55. Gene product 55 (gp55) confers specificity for late T4 promoters on RNA polymerase core (26). The roles of gp33 and gp45 in transcription remain unclear, but gp45 is known also to play a role in DNA replication as an accessory protein to the replication complex (1). Late promoters share the sequence TATAAATA centered at about position -10 from the start of transcription, but require no position -35 sequence (12,14).Late transcription is also distinguished by a requirement for concurrent viral DNA replication (45). Late transcription can be uncoupled from replication in genetic backgrounds which are thought to stabilize nicks in the DNA. These observations led to the hypothesis that late transcription requires a competent template which has been activated by DNA replication (46). Uncoupling can also be achieved by placing the promoter on a plasmid template (21, 23). Late transcription from plasmid templates in vivo is otherwise faithful in its initiation sites and in regulation by genes 33, 45, and 55 (14, 23). Additionally, supercoiled plasmids are efficient templates for in vitro transcription with purified T4 late RNA polymerase (24). Therefore it appears that plasmid templates are intrinsically competent for late transcription.The topoisomer profile of plasmids changes drastically after T4 infection. We have argued that there are two reasons for this topoisomerizat...

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Section: Discussionsupporting

confidence: 76%

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

1988

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“…Blocking DNA synthesis limits transcription mostly to this gene class (19). In the experiments reported here, cells were infected with phage of the various rIIB genotypes containing amber mutations in genes 32 and 44, the products of which are required for phage DNA synthesis (10).…”

Section: Resultsmentioning

confidence: 99%

Temperature-sensitive mutation in the initiation codon of the rIIB gene of bacteriophage T4.

Belin¹,

Hedgpeth²,

Selzer³

et al. 1979

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

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We have determined the sequence of a ribosome-protected region of T4 rI1B mRNA labeled in vivo. The rI1B mutant HD263, which is temperature sensitive for translation, has an altered initiation codon (AUA instead of AUG). Because wild-type and HD263 proteins have the same fMet peptide, this AUA is used to initiate translation in the mutant HIB mRNA. We have also identified a six-base sequence which is complementary to the 3' end of the 16S rRNA. Most temperature-sensitive mutations exert their effect on the function or stability of a protein via a change in amino acid sequence. However, some mutations called tss (for temperature-sensitive synthesis) affect the synthesis of the protein rather than its function (1). A priori, a tss mutation could affect transcription of the mutant gene, processing or translation of the mutant mRNA, or assembly of the mutant protein into a correct quaternary structure.Bacteriophage T4 strain HD263 has a temperature-sensitive mutation located at the very beginning of gene rIIB (2). It is thought to be a tss mutation that affects translation of the rIIB mRNA because of the following observations: (i) Infections with the mutant at 370C, or above, produce no detectable rIIB protein, whereas at 25°C the rate of synthesis of rIIB protein is 10-15% of that observed in rIIB+ infections (3, 4). (ii) A tryptic peptide analysis of the HD263 and wild-type rIIB proteins has failed to reveal any difference between the two proteins (5). (iii) Reduced transcription, mRNA inactivation, or protein degradation cannot account for the mutant phenotype (3, 4). (iv) The synthesis of rIIB protein directed by RNA extracted from HD263-infected cells is temperature sensitive in vitro, especially when ribosomes or initiation factors are limiting (4).These observations, together with the map location of the mutation, suggested that HD263 affects some early step in the translation of the rIIB mRNA-for example, the binding of the ribosome to the mRNA or the alignment of the fMet-tRNA at the initiation codon. Attachment of the ribosome involves a transient base-pairing between nucleotides near the 3' end of the 16S rRNA and a purine-rich sequence in the untranslated part of the mRNA (6, 7). Alignment of the fMet-tRNA requires base pairing between the anticodon of the tRNA and the initiation codon of the mRNA (8). An extensive discussion of events involved in translation initiation has been published (9).In this communication we report the sequence of a 25-nucleotide segment of the rIIB mRNA that contains a sequence complementary to the 16S RNA and the first three codons of gene rIIB. We have found that the wild-type initiation codon AUG is changed to AUA in mutant HD263. Since both mutant and wild-type rIIB proteins have the same fMet peptide (5), an AUA codon can therefore be used to initiate translation in phage-infected cells, at least at 250C. MATERIALS AND METHODSBacteriophage. The phage of the various rIIB genotypes contained mutations 32amH18 and 44amN82 (10). Phage stocks grown on Escherichia coli ...

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“…For prokaryotes, gene activity can be regulated by proteins binding to specific regulatory DNA sequences (1)(2)(3)(4)(5)(6). In eukaryotes, the mechanisms of gene regulation are unknown, but we can assume that a sequence-specific interaction between regulatory molecules and the DNA is a prerequisite for regulation at the transcriptional level.…”

mentioning

confidence: 99%

Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.

Weideli¹,

Brack²,

Gehring³

1980

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

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A DNA-binding protein (DB-2) was isolated from unfertilized Drosophila eggs by DNA-cellulose chromatography. In competition assays with DNA from other species, DB-2 preferentially binds to Drosophila DNA. This binding protein can also be isolated from pupal nuclei and comprises only a small fraction (<0.01%) of the total nonhistone chromosomal proteins. In order to investigate the specificity of the interaction between DB-2 and the DNA, we attempted to isolate the DNA sequences to which DB-2 binds. DB-2 was used as a probe to screen our gene bank established by inserting randomly sheared fragments of Drosophila DNA into bacterial plasmids. Groups of plasmids were tested for binding to DB-2 by a filter binding assay. The plasmids bound to the nitrocellulose filter were eluted and used for bacterial transformation. After several cycles of transformation and cloning, two plasmids, A17 and BlO, were isolated that bind DB-2 specifically, as measured by filter binding and competition assays. In plasmid A17, binding of DB-2 protects two short DNA segments of approximately 13 and 30 base pairs from digestion by DNase I. By filter hybridization according to Southern, these sequences were mapped to a defined restriction fragment. Further evidence for the binding specificity was obtained by visualizing the protein-DNA complex in the electron microscope. In salivary gland giant chromosomes, A17 DNA hybridizes to a single site (95A/B) on chromosome 3.For prokaryotes, gene activity can be regulated by proteins binding to specific regulatory DNA sequences (1-6). In eukaryotes, the mechanisms of gene regulation are unknown, but we can assume that a sequence-specific interaction between regulatory molecules and the DNA is a prerequisite for regulation at the transcriptional level. Based upon this assumption, several studies have concentrated on the isolation of proteins associated with specific fractions of DNA or chromatin. Proteins associated with inactive chromatin (7) and a protein binding preferentially to a DNA satellite (8) have been purified. By use of fluorescent antibody techniques, the binding of specific proteins to a limited number of chromosomal loci or puffs in polytene chromosomes has been demonstrated (9, 10). In our laboratory, a DNA-binding protein, DB-1, has been purified which binds specifically to a cloned segment of nucleolar DNA in Drosophila (11). In this study, we describe the isolation of another DNA-binding protein, DB-2, the cloning of the DNA sequences to which it binds, and the sequence specificity of the DNA-protein interaction. METHODSPurification of DB-2. The isolation of total DNA-binding proteins from unfertilized eggs and from pupal nuclei of Drosophila melanogaster by DNA-cellulose chromatography was described earlier (11). Protein DB-2 was purified from total DNA-binding proteins by Sephadex-gel filtration and preparative NaDodSO4/polyacrylamide gel electrophoresis and separated from DB-1 by isoelectric focusing in the presence of 6 M urea essentially as described for protein DB-1 (...

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Controls and polarity of transcription during bacteriophage T4 development

Cited by 85 publications

References 34 publications

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

Temperature-sensitive mutation in the initiation codon of the rIIB gene of bacteriophage T4.

Characterization of Drosophila DNA-binding protein DB-2: demonstration of its sequence-specific interaction with DNA.

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