A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme

Abstract

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...

The bacteriophage T4 AsiA protein contacts the β-flap domain of RNA polymerase

“…In prior work we demonstrated that the ability of AsiA to stably associate with the RNAP holoenzyme and inhibit 70 -dependent transcription depends on the strength of the 70 region 4/␤-flap interaction (23). Thus, we showed that substitutions in 70 region 4 that weaken the 70 region 4/␤-flap interaction facilitate AsiAdependent transcription inhibition and substitutions that strengthen the 70 region 4/␤-flap interaction have the opposite effect.…”

“…To do this, we used either a CI-␤-flap fusion protein lacking the ␤-flap-tip helix or an ␣-70 fusion protein carrying amino acid substitution F563Y, which weakens the 70 region 4/AsiA interaction (23). We found that disrupting either the AsiA/␤-flap interaction or the 70 region 4/AsiA interaction abolished the stimulatory effect of AsiA on lacZ transcription (Fig.…”

“…Two mechanistic consequences of the AsiA/ 70 region 4 interaction have been described. First, the interaction occludes determinants of 70 region 4 that are required for the 70 region 4/␤-flap interaction (22,23). Second, AsiA stabilizes an alternative conformation of region 4 in which its DNA-binding surface is deformed (24).…”

“…1A). We previously used this 2-hybrid assay to study both the 70 region 4/␤-flap interaction (28) and the 70 region 4/AsiA interaction (23,29). To assay the ability of AsiA to interact with the ␤-flap, we used a CI-AsiA fusion protein and an ␣-␤-flap fusion protein (bearing the ␤-flap in place of the C-terminal domain of ␣).…”

Dissection of the Bacteriophage T4 Late Promoter Complex

“…An additional surprising result was finding that T7A1 DNA, heparin and RNAP came to equilibrium and reached the same final state relatively quickly, regardless of order of mixing, under the conditions of this experiment. The ability to form transcription complexes in the face of competition by heparin was lost when the σ 70 domain 4-β flap interaction was disrupted by mutations R541C/L607P (Gregory et al, 2004), and diminished when σ 70 lacked its N-terminal domain 1.1 ( Figure 6B). Thus, a parallel can be drawn between these σ 70 and gp55 promoters: sufficiently avid formation of promoter complexes to overcome the heparin competitor characterized those promoter complexes with the more extensive set of protein-protein as well as protein-DNA interactions and was sensitive to loss of these individual interactions.…”