The Escherichia coli RNA polymerase.anti-sigma 70 AsiA complex utilizes alpha-carboxyl-terminal domain upstream promoter contacts to transcribe from a -10/-35 promoter.
During infection of Escherichia coli, the phage T4 early protein AsiA inhibits open complex formation by the RNA polymerase holoenzyme Efinal sigma(70) at -10/-35 bacterial promoters through binding to region 4.2 of the final sigma(70) subunit. We used the -10/-35 lacUV5 promoter to study the properties of the Efinal sigma(70). AsiA complex in the presence of the glutamate anion. Under these experimental conditions, inhibition by AsiA was significantly decreased. KMnO(4) probing showed that the observed residual transcriptional activity was due to the slow transformation of the ternary complex Efinal sigma(70). AsiA.lacUV5 into an open complex. In agreement with this observation, affinity of the enzyme for the promoter was 10-fold lower in the ternary complex than in the binary complex Efinal sigma(70).lacUV5. A tau plot analysis of abortive transcription reactions showed that AsiA binding to Efinal sigma(70) resulted in a 120-fold decrease in the second-order on-rate constant of the reaction of Efinal sigma(70) with lacUV5 and a 55-fold decrease in the rate constant of the isomerization step leading to the open complex. This ternary complex still responded to activation by the cAMP.catabolite activator protein complex. We show that compensatory Efinal sigma(70)/promoter upstream contacts involving the C-terminal domains of alpha subunits in Efinal sigma(70) become essential for the binding of Efinal sigma(70). AsiA to the lacUV5 promoter.
Orsini
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G
Unité de Physico-Chimie des Macromolécules Biologiques, CNRS URA 1773, Département de Biologie Moléculaire, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Kolb
A
A
Buc
H
H
D001483:Base Sequence; D018983:DNA Footprinting; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011196:Potassium Permanganate / Q000737:chemistry; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D017931:DNA Primers; D014764:Viral Proteins; D011196:Potassium Permanganate; D012321:DNA-Directed RNA Polymerases