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The bacteriophage T4 middle promoter P uvsX : analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription
Author(s) -
MarchAmegadzie Roslyn,
Hinton Deborah M.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.tb02374.x
Subject(s) - biology , transcription (linguistics) , promoter , rna polymerase , footprinting , microbiology and biotechnology , bacteriophage , dna footprinting , dna , consensus sequence , polymerase , activator (genetics) , transcription factor , genetics , gene , rna , gene expression , escherichia coli , philosophy , linguistics , base sequence
Summary Bacteriophage T4 middle promoters, which are transcribed using phage‐modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host σ 70 consensus sequence at – 10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at – 30. While the T4 middle promoter P uvsx has these – 10 and –30 motifs, it also has matches to the MotA box at –35, –51, –70, and –87. We show that MotA binds to P uvsx DNA, footprinting a region that includes the MotA boxes at –30, –35, and –51. Very high levels of MotA are required for footprinting and gel‐shift experiments, and protein‐DNA complexes formed in the presence of both phage‐modified polymerase and MotA are more resistant to Hindlll cleavage than those formed with either protein alone. These results suggest that MotA‐DNA interactions may be stabilized by phage‐modified polymerase. Sequences between –18 and –38 are absolutely required for MotA activation of transcription, but sequences upstream of –38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect P uvsx into a core promoter, downstream of ‐38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein‐DNA interactions.