The 2.6 A crystal structure of a fragment of the sigma 70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the -10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.

The structure of E. coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals. The structure reveals extensive conformational changes when compared with the previously determined E. coli RNAP holoenzyme structure, but resembles the yeast RNAPII structure. While each of these structures contains a thumb-like projection surrounding a channel 25 A in diameter, the E. coli RNAP holoenzyme thumb defines a deep but open groove on the molecule, whereas the thumb of E. coli core and yeast RNAPII form part of a ring that surrounds the channel. This may define promoter-binding and elongation conformations of RNAP, as E. coli holoenzyme recognizes promoter sites on double-stranded DNA, while both E. coli core and yeast RNAPII are elongating forms of the polymerase and are incapable of promoter recognition.

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The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the ␤ subunit and conserved region 4 of the subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the region 4͞␤-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the ␤-flap for binding to region 4, thereby inhibiting transcription initiation by disrupting the region 4͞␤-flap interaction.T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically proficient core enzyme (subunit structure ␣ 2 ␤␤Ј ) and a subunit that confers on the holoenzyme the ability to recognize specific promoter sequences (1). The primary factor in Escherichia coli is 70 , and the 70 -containing holoenzyme (E 70 ) typically recognizes promoters defined by two conserved sequence elements (the Ϫ10 and Ϫ35 hexamers) positioned roughly 10 and 35 bp upstream of the transcription start point (1). Most factors share four conserved regions (2), and of these, regions 2 and 4 interact with the Ϫ10 and Ϫ35 elements, respectively (1). Nevertheless, intact 70 recognizes specific promoter sequences only in the context of the holoenzyme because of critical conformational changes in 70 that take place when it associates with the core enzyme (3). These include an increase in the interdomain distance between regions 2 and 4 that is caused by an interaction between region 4 and the ␤-flap domain (4). The region 4͞␤-flap interaction is essential for the recognition of Ϫ10͞Ϫ35 promoters because it positions regions 2 and 4 of 70 for simultaneous interaction with the promoter Ϫ10 and Ϫ35 elements (4). This role of the region 4͞␤-flap interaction suggested that there may exist regulatory factors that inhibit transcription from Ϫ10͞Ϫ35 promoters by disrupting the region 4͞␤-flap interaction (4, 5).Here we examine the mechanism of action of the bacteriophage T4-encoded anti-factor AsiA. AsiA is known to bind tightly to region 4 of 70 and inhibit transcription from Ϫ10͞Ϫ35 promoters when complexed with the holoenzyme (6-10). The finding that AsiA binds to region 4 of 70 and inhibits transcription specifically from Ϫ10͞Ϫ35 promoters raises the possibility that AsiA works by disrupting the 70 region 4͞␤-flap interaction, a model that is consistent with recent NMR analysis (11). We provide genetic evidence that AsiA and the ␤-flap interact with overlapping determinants on 70 region 4. We then present complementary biochemical and biophysical evidence that AsiA works by a competitive binding mechanism, inhibiting transcription from Ϫ10͞Ϫ35 promoters by disrupting the region 4͞␤-flap interaction in the context of the holoenzyme. Materials and MethodsMutant Screen. Mutagenic PCR was u...