The Escherichia coli RNA Polymerase·Anti-ς70 AsiA Complex Utilizes α-Carboxyl-terminal Domain Upstream Promoter Contacts to Transcribe from a −10/−35 Promoter

Gilbert Orsini,Annie Kolb,Henri Buc

doi:10.1074/jbc.m010105200

Abstract

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.

Highlights

In Escherichia coli RNA polymerase, the core enzyme E associates with the ␴ subunit to form the holoenzyme E␴, the species able to initiate transcription at specific promoter sites on bacterial or phage genomes
We describe the behavior of the E␴701⁄7AsiA complex in the presence of KCl and KGlu, two salts widely used in transcription studies
By runoff transcription analysis on the lacUV5 promoter, we show that the species E␴701⁄7AsiA is much less inhibited in KGlu than in KCl (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—High Pure spin columns and the Pwo polymerase were from Roche Molecular Biochemicals (Mannheim, Germany). RNA polymerase holoenzyme (40 pmol) and an 8-fold molar excess of AsiA were incubated in a 30-␮l reaction for 30 min at 37 °C in buffer A containing 100 mM KGlu and 0.125% (v/v) Tween instead of bovine serum albumin. DNase I Footprinting—Complexes with the labeled lacUV5 promoter (at a 3 nM final concentration) were formed during 50 min at 37 °C in buffer A containing 200 mM KGlu or 100 mM KCl, using a 6-fold molar excess of AsiA or the cAMP1⁄7CAP complex as indicated, with purified RNA polymerase or reconstituted ⌬␣-235 RNA polymerase (each at a 100 nM final concentration). Protected bands were identified on the pattern afforded by the migration of the same fragment treated for the G ϩ A sequencing reaction (29)

RESULTS

Role of Upstream Contacts in the Properties of the Ternary

Holoenzyme form kf KBkf

DISCUSSION