S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression

Hattman, Stanley; Ives, Janet

doi:10.1016/0378-1119(84)90179-3

Cited by 32 publications

(21 citation statements)

References 19 publications

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“…In addition to the overlap between the N and P transcripts, there is also an 8-bp overlap between the N and P coding regions (22). Similarly, the promoter-distal portion of the Mu gin transcript overlaps at least 80 bases of the 5' end of the mom transcript (28,61), and there is translational overlap between the com and mom coding regions (40). In addition to these examples in Mu, there are other instances in which phage and host promoters are located within coding regions; for example, the G0o99 and Go 06promoters of phage M13 are located in the upstream gene II (43), the gene B promoter in phage 4gX174 is located within the upstream gene A (65), in E. coli the infC promoter is situated at least 175 bp upstream from the 3' end of the thrS gene (63) and a promoter for the E. coli signal peptidase gene Isp lies well within the upstream ileS gene (57).…”

Section: Resultsmentioning

confidence: 99%

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

1989

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Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. Si nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli C70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent lI-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.Viruses have evolved a number of regulatory schemes to ensure efficient production of progeny particles during lytic development. A common strategy is to sequentially activate and inhibit transcription of different sets of viral genes. In general, genes for DNA replication are transcribed early, while those for'virion formation and release are expressed later. Activation of new sets of genes often involves utilization of new promoter sequences which are recognized by new virus-specific polymerases or by altered forms of the host polymerase. For example, Escherichia coli phages T7 and N4 synthesize their own RNA polyrmerases (12, 18), whereas Bacillus phage SPOl and E. coli phage T4 produce phage-encoded sigma factors which substitute for the host sigma subunit (14, 42). E. coli phage lambda uses two additional mechanisms: it produces accessory DNA-binding proteins, cI and cII, which enhance initiation by host RNA polymerase holoenzyme at specific promoters (55,69), and it modifies the host RNA polymerase complex with phage proteins N and Q to achieve antitermination and thus elongation of short constitutive leader transcripts (1,19

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Section: Resultsmentioning

confidence: 99%

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

1989

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“…Asterisks indicate the three GATC sites in region I. Positions 974 and 963 indicate the different mRNA starts that were found by S1 nuclease mapping (9,33). The mom transcript is indicated by a broken line; the arrow marks the direction of transcription.…”

Section: Methodsmentioning

confidence: 99%

“…Current models suggest that an Escherichia coli protein may act as a repressor, turning off morm gene transcription by. binding to region I when the GATC sites are un-or hemimethylated; this in turn could prevent access of C or RNA polymerase (9). For a more direct understanding of protein-DNA interactions in the regulation of mom gene transcription, characterization of individual components is necessary.…”

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confidence: 99%

Role of bacteriophage Mu C protein in activation of the mom gene promoter

1989

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The phage Mu C gene product is a specific activator of Mu late gene transcription, including activation of the mom operon. Fusion of the C gene to the efficient translation initiation region of the Escherichia coli atpE gene allowed significant overproduction of C protein, which was subsequently purified and assayed for DNA binding by gel retardation and nuclense footprinting techniques. C protein binds to a site immediately upstream of the -35 region both of the mom promoter and the related phage D108 mod promoter. The location of the mom promoter has been determined by primer extension. Upstream deletions extending more than 3 base pairs into the C-binding site abolished activation of the mom promoter in vivo. In vitro binding of C was not significantly affected by DNA methylation. A second, C-dependent promoter was identified just downstream of the C coding region; comparison with the mom promoter revealed common structural elements.

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“…transcription phase is a good example of one such regulatory scheme. Although mom gene expression seems to be dispensable for Mu growth or lysogeny, premature activation or constitutive expression of mom function is detrimental to the host cells (49,50). Hence, it is not surprising that intricate mechanisms have been evolved for the regulation of mom expression at both the transcriptional and translational levels (16).…”

Section: Dna Structure Analysis Of Tmentioning

confidence: 99%

Intrinsic DNA Distortion of the Bacteriophage MumomP1 Promoter Is a Negative Regulator of Its Transcription

Basak

Olsen

Hattman

et al. 2001

Journal of Biological Chemistry

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The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19-base pair suboptimal spacer between the ؊35 (ACCACA) and ؊10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its own. DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5 end of the ؊10 element might prevent RNA polymerase from binding to momP1. To investigate the influence of the T 6 run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline ((OP) 2 Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription. This is a novel mechanism of regulation of toxic gene expression.

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S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression

Cited by 32 publications

References 19 publications

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

Role of bacteriophage Mu C protein in activation of the mom gene promoter

Intrinsic DNA Distortion of the Bacteriophage MumomP1 Promoter Is a Negative Regulator of Its Transcription

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