Unusual interaction of RNA polymerase with the bacteriophage Mu middle promoter Pm in the absence of its activator protein Mor

MicrobiologyOpen

Yongkai Mo¹ 

[1] Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, 38163

关键词: Bacteriophage Mu;    Mor activator protein;    Mu middle promoter Pm;    prokaryotic transcription;    RNAP‐promoter interactions;   

DOI  :  10.1002/mbo3.181

来源: Wiley

【 摘 要 】

Abstract

The bacteriophage Mu Mor activator protein is absolutely required for transcription from the Mu middle promoter P_m. However, when RNA polymerase (RNAP) was incubated with P_m DNA in the absence of Mor, a band at promoter position −51 was hypersensitive to DNase I cleavage, demonstrating an interaction of RNAP with the promoter DNA. The hypersensitivity was similar at four different lengths of P_m DNA assayed from −62 to +10, −62 to +46, −96 to +10, and −96 to +46. The hypersensitivity occurred equally well at 5°C, 15°C, and 30°C, indicating that it did not require open complex formation, which only occurred at 30°C. The −51 hypersensitivity at 5°C and 15°C was eliminated by the addition of heparin, consistent with the possibility that it arose by formation of unstable closed complexes of RNAP bound to P_m DNA. Generation of the hypersensitive band required the complete RNAP with its αCTDs, but neither the αCTD nor intact α were sufficient for the interaction and resulting hypersensitivity. There was no correlation between the level of hypersensitivity observed in vitro and the level of P_m activity in vivo, as assayed by the Mor-dependent production of β-galactosidase from a P_m-lacZ fusion. In an “order of addition” experiment, preincubation of P_m DNA with Mor followed by addition of RNAP led to the fastest open complex formation, whereas preincubation of P_m DNA with RNAP gave the slowest. These results support the conclusion that Mor recruits RNAP to P_m rather than reposition a prebound RNAP, as occurs for C-dependent repositioning of RNAP at the Mu late promoter P_mom.

【 授权许可】

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© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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