The interaction between the AsiA protein of bacteriophage T4 and the sigma70 subunit of Escherichia coli RNA polymerase.
The AsiA protein of bacteriophage T4 binds to the sigma70 subunit of Escherichia coli RNA polymerase and plays a dual regulatory role during T4 development: (i) inhibition of host and phage early transcription, and (ii) coactivation of phage middle-mode transcription, which also requires the T4 DNA binding transcriptional activator, MotA. We report that the interaction between AsiA and sigma70 occurs with a 1:1 stoichiometry. When preincubated with RNA polymerase, AsiA is a potent inhibitor of open complex formation at the lac UV5 promoter, whereas it does not perturb preformed open or intermediate promoter complexes. DNase I footprinting and electrophoretic mobility shift analyses of RNA polymerase-DNA complexes formed at the T4 early promoter P15.0 show that AsiA blocks the initial RNA polymerase binding step that leads to the formation of specific closed promoter complexes. A contrasting result is obtained on the T4 middle promoter PrIIB2, where AsiA stimulates the formation of both closed complexes and open complexes. Therefore, we propose that AsiA modulates initial DNA binding by the RNA polymerase, switching promoter usage at the level of closed complex formation.
Adelman
K
K
Centre de Génétique Moléculaire du CNRS, UPR 9061, Laboratoire Associé à l'Université Pierre et Marie Curie, 91198 Gif-sur-Yvette Cedex, France.
Orsini
G
G
Kolb
A
A
Graziani
L
L
Brody
E N
EN
D001426:Bacterial Proteins / Q000378:metabolism; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012808:Sigma Factor / Q000302:isolation & purification / Q000378:metabolism*; D013604:T-Phages / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I