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J. Biol. Chem., Vol. 276, Issue 23, 19812-19819, June 8, 2001

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The Escherichia coli RNA Polymerase·Anti-⁷⁰ AsiA Complex Utilizes -Carboxyl-terminal Domain Upstream Promoter Contacts to Transcribe from a 10/35 Promoter^*

Gilbert Orsini, Annie Kolb, and Henri Buc

From the 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

During infection of Escherichia coli, the phage T4 early protein AsiA inhibits open complex formation by the RNA polymerase holoenzyme E⁷⁰ at 10/35 bacterial promoters through binding to region 4.2 of the ⁷⁰ subunit. We used the 10/35 lacUV5 promoter to study the properties of the E⁷⁰·AsiA complex in the presence of the glutamate anion. Under these experimental conditions, inhibition by AsiA was significantly decreased. KMnO₄ probing showed that the observed residual transcriptional activity was due to the slow transformation of the ternary complex E⁷⁰·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 E⁷⁰·lacUV5. A tau plot analysis of abortive transcription reactions showed that AsiA binding to E⁷⁰ resulted in a 120-fold decrease in the second-order on-rate constant of the reaction of E⁷⁰ 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 E⁷⁰/promoter upstream contacts involving the C-terminal domains of  subunits in E⁷⁰ become essential for the binding of E⁷⁰·AsiA to the lacUV5 promoter.

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