Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase

Abstract

Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters. To address its mechanism of action, we overproduced the -16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site. A protein fraction highly enriched for Escherichia coli RNA polymerase (Eo70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Pac) and a Mu late promoter, PIYS. The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical. When anti-o70 antibody was added to C-dependent transcription reactions containing both Pt,., and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess Eo70. This result suggests that C-dependent activation of P, requires 70.Further supporting evidence was provided by a reconstitution experiment in which an E 70-depleted fraction containing C was unable to activate transcription from P,y unless both purified r70 and core polymerase were added. These results strongly suggest that C is not a new sigma factor but acts as an activator for Eo70-dependent transcription.

Identification of a positive regulator of the Mu middle operon

“…Late transcription begins 10 to 12 min after induction and increases until lysis at 45 to 60 min (36,50,59). The Mu C gene product serves as an activator for E. coli RNA polymerase-dependent transcription from four late promoters:Pjs, PI, Pp., and Pmom (5,34,35,50). These late transcripts encode the genes involved in phage morphogenesis, DNA modification, and cell lysis (5,24,26,36,50,59 (50, 51).…”

“…Pjs, PI, Pp., and Pmom (5,34,35,50). These late transcripts encode the genes involved in phage morphogenesis, DNA modification, and cell lysis (5,24,26,36,50,59 (50, 51).…”

“…After 20 min of incubation for mRNA turnover, the cells were pulsed for 5 min with 10 ,uCi of [35S]methionine (1,200 Ci/mmol). The samples were then boiled in cracking buffer and subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (34). The proteins were detected by autoradiography (34).…”

“…The plasmid pWM6, aPvul-SalI deletion derivative of pKN50, contains a 5,639-bp EcoRIPvuI fragment of Mu DNA (Mu coordinates 5.1 through 10.7 [33]). The plasmid pWM15 is a pGEM-3Z derivative containing a 515-bp HpaI-XmnI fragment of Mu DNA (Mu coordinates 9.946 through 10.461 [34]) that expresses the C protein.…”

Dual Regulatory Control of a Particle Maturation Function of Bacteriophage P1

“…Complex regulatory networks have been elucidated for phages like T4, , P2/P4, Mu, and T7, among others. The investigations of various regulatory phage proteins like antiterminators (9,32), repressors (18,35), activators (1,19,26,36), sigma factors (8), antisigma factors (15), and RNA polymerases (27) provided major contributions to our understanding of principal regulatory concepts.…”