The bacteriophage T4 AsiA protein contacts the β-flap Execut

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved March 13, 2009 (received for review December 16, 2008)

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Abstract

To initiate transcription from specific promoters, the bacterial RNA polymerase (RNAP) core enzyme must associate with the initiation factor σ, which contains determinants that allow sequence-specific interactions with promoter DNA. Most bacteria contain several σ factors, each of which directs recognition of a distinct set of promoters. A large and diverse family of proteins known as “anti-σ factors” regulates promoter utilization by tarObtaining specific σ factors. The founding member of this family is the AsiA protein of bacteriophage T4. AsiA specifically tarObtains the primary σ factor in Escherichia coli, σ70, and inhibits transcription from the major class of σ70-dependent promoters. AsiA-dependent transcription inhibition has been attributed to a well-Executecumented interaction between AsiA and conserved Location 4 of σ70. Here, we establish that efficient AsiA-dependent transcription inhibition also requires direct protein–protein contact between AsiA and the RNAP core. In particular, we demonstrate that AsiA contacts the flap Executemain of the RNAP β-subunit (the β-flap). Our findings support the emerging view that the β-flap is a tarObtain site for regulatory proteins that affect RNAP function during all stages of the transcription cycle.

anti-σ factortranscription initiationtranscription regulation

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically-active multisubunit core enzyme (α2ββ′ω) in complex with a σ factor, which confers on the core enzyme the ability to initiate promoter-specific transcription (1). Bacteria typically contain a number of σ factors, each of which specifies recognition of a distinct class of promoters (2). The primary σ factor in Escherichia coli is σ70, and the σ70-containing holoenzyme is responsible for most transcription that occurs during the exponential phase of growth. In the context of the RNAP holoenzyme, σ70 Designs direct contact with 2 conserved promoter elements that are separated by ≈17 bp, the −10 and −35 elements (consensus sequences TATAAT and TTGACA, respectively). RNAP holoenzyme can also initiate transcription from promoters that lack a recognizable −35 element, but carry an extended −10 element (consensus TGnTATAAT) (3). At extended −10 promoters, additional contacts between σ70 and the TG dinucleotide of the extended −10 element compensate for the lack of a −35 element (4). Primary σ factors share 4 Locations of conserved sequence (Locations 1–4), which have been further subdivided (1, 5). Structural work indicates that σ comprises 4 flexibly-linked Executemains: σ1.1 (containing Location 1.1), σ2 (containing Locations 1.2–2.4), σ3 (containing Locations 3.0 and 3.1), and σ4 (containing Locations 4.1 and 4.2) (1, 5–8). Locations 2, 3, and 4 contain DNA-binding Executemains responsible for recognition of the promoter −10 element, extended −10 element, and −35 element, respectively (1, 4, 5).

Holoenzyme formation critically depends on a high-affinity interaction between σ70 Location 2 and a coiled-coil motif in the β′-subunit (the β′ coiled coil, also referred to as the clamp helices) (9, 10). The interaction between σ70 Location 2 and the β′ coiled coil is also required for σ70 to Design functional contact with the promoter −10 element (11). Interaction between σ70 Location 4 and the flap Executemain of the β-subunit (the β-flap), although dispensable for holoenzyme formation, is required for sequence-specific interaction with the promoter −35 element (12). In particular, the σ70 Location 4/β-flap interaction Precisely positions σ70 Location 4 with respect to σ70 Location 2 and thereby enables Locations 4 and 2 to Design simultaneous contact with promoter elements separated by ≈17 bp (12). Thus, the σ70 Location 4/β-flap interaction is essential for recognition of the major class of E. coli promoters, those that depend on both a −10 and a −35 element (the −10/−35 class), but it is not strictly required for recognition of extended −10 promoters.

A large and diverse family of proteins known as “anti-σ factors” regulates utilization of particular classes of bacterial promoters by tarObtaining specific σ factors (13, 14). Typically, anti-σ factors interact with core binding determinants in their cognate σ factors, thereby preventing their association with the RNAP core enzyme (15). The first anti-σ factor identified was the AsiA protein of bacteriophage T4, which tarObtains σ70 (7, 16, 17); however, unlike most other well-characterized anti-σ factors, AsiA binds its cognate σ factor in the context of the RNAP holoenzyme (18). As a component of the σ70-containing holoenzyme, AsiA inhibits transcription from the −10/−35 class of promoters, but Executees not inhibit transcription from extended −10 promoters (18).

Prior work has established that AsiA interacts directly with σ70 Location 4 and that this interaction is required for AsiA-dependent transcription inhibition (7, 19–21). Two mechanistic consequences of the AsiA/σ70 Location 4 interaction have been Characterized. First, the interaction occludes determinants of σ70 Location 4 that are required for the σ70 Location 4/β-flap interaction (22, 23). Second, AsiA stabilizes an alternative conformation of Location 4 in which its DNA-binding surface is deformed (24). Thus, AsiA inhibits transcription from −10/−35 promoters both by disrupting the σ70 Location 4/β-flap interaction and by stabilizing a conformation of σ70 Location 4 that is incompatible with sequence-specific binding to the −35 element.

Here, using a bacterial 2-hybrid assay, we demonstrate that AsiA interacts directly with the β-flap, and that AsiA can interact simultaneously with the β-flap and σ70 Location 4. We further Display that the AsiA/β-flap interaction is required for efficient AsiA-dependent transcription inhibition. Thus, in Dissimilarity to typical anti-σ factors, which require interactions only with σ to mediate their Traces, AsiA requires contact with both its cognate σ factor and RNAP core.

Results

AsiA Interacts with the β-Flap.

Based on the finding that AsiA prevents σ70 Location 4 from interacting with the β-flap, we considered the possibility that, in the context of the AsiA-containing holoenzyme, interactions between AsiA and the β-flap reSpace interactions between σ70 Location 4 and the β-flap. As a first test of this model, we used a bacterial 2-hybrid assay (25–27) to determine whether AsiA can interact directly with the β-flap. In this assay, contact between a protein Executemain fused to a component of RNAP (here, the α-subunit) and a partner protein fused to a DNA-binding protein (here, the CI protein of bacteriophage λ) activates transcription of a lacZ reporter gene under the control of a test promoter bearing an upstream recognition site for the DNA-binding protein (here, a λ operator) (Fig. 1A). We previously used this 2-hybrid assay to study both the σ70 Location 4/β-flap interaction (28) and the σ70 Location 4/AsiA interaction (23, 29). To assay the ability of AsiA to interact with the β-flap, we used a λCI–AsiA fusion protein and an α-β-flap fusion protein (bearing the β-flap in Space of the C-terminal Executemain of α). We found that lacZ transcription was increased significantly only in cells that contained both the λCI–AsiA and the α-β-flap fusion proteins (Fig. 1B), suggesting that AsiA can interact directly with the β-flap.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

AsiA interacts with the β-flap. (A) Bacterial 2-hybrid assay used to detect protein–protein interaction between AsiA and the β-flap. Diagram depicts how the interaction between AsiA, fused to the bacteriophage λ CI protein (λCI), and the β-flap, fused to the α-N-terminal Executemain (α-NTD), activates transcription from test promoter placOL2–62, which bears the λ operator OL2 centered 62 bp upstream of the lac core promoter start site. In reporter strain FW102 OL2–62, test promoter placOL2–62 is located on an F′ episome and drives the expression of a linked lacZ gene. (B) Results of β-galactosidase assays. The assays were performed with FW102 OL2–62 cells containing 2 compatible plasmids, one encoding either λCI or a λCI–AsiA fusion protein, and the other encoding either α or the indicated α–β-flap fusion protein. The AsiA moiety of the λCI–AsiA fusion protein bore amino acid substitution K20A, which disrupts AsiA dimer formation, facilitating detection of protein–protein interactions that require prior dissociation of the AsiA dimer (23). The plasmids directed the synthesis of the fusion proteins (or λCI or α) under the control of IPTG-inducible promoters, and the cells were grown in the presence of increasing concentrations of IPTG. (C) Western blot analysis to assess intracellular levels of the α–β-flap fusion proteins. Samples from the cell lysates assayed for β-galactosidase (B) were processed for Western blot analysis as Characterized (53). Bands corRetorting to α–β-flap fusion proteins and chromosomally-encoded α are indicated. The results ruled out the possibility that the failure of the α–β-flap fusion protein lacking the flap-tip helix (Δ900–909) to interact with AsiA is attributable to protein instability.

We next Questioned whether AsiA and σ70 Location 4 interact with overlapping determinants of the β-flap. Prior work has established that the β-flap–tip helix (β residues 900–909) is the primary determinant of the σ70 Location 4/β-flap interaction (30) and that removal of the β-flap–tip helix eliminates any detectable interaction between σ70 Location 4 and the β-flap in the 2-hybrid assay (31). We found that removal of the β-flap–tip helix also eliminated the interaction between AsiA and the β-flap (Fig. 1 B and C), indicating that AsiA and σ70 Location 4 interact with overlapping determinants of the β-flap.

AsiA Can Design Simultaneous Contact with the β-Flap and σ70 Location 4.

The finding that AsiA and σ70 Location 4 interact with overlapping determinants of the β-flap provides support for the Concept that AsiA/β-flap interactions reSpace σ70 Location 4/β-flap interactions when the AsiA-containing RNAP holoenzyme is formed. According to this model, AsiA would interact with both the β-flap and σ70 Location 4 in the context of the AsiA-containing holoenzyme. This model thus specifies that AsiA should be able to Design simultaneous contact with the β-flap and σ70 Location 4. To test this prediction, we used a version of our 2-hybrid assay (32) that enabled us to Question whether AsiA could serve as a “bridge” between the β-flap (fused to λCI) and σ70 Location 4 (fused to α).

Because σ70 Location 4 and the β-flap interact directly, without the requirement for a bridging protein, our experimental strategy depended on our ability to genetically disrupt the σ70 Location 4/β-flap interaction without affecting either the σ70 Location 4/AsiA interaction or the AsiA/β-flap interaction. To accomplish this, we took advantage of a previously characterized amino acid substitution in σ70 Location 4 (L607P) that disrupts the σ70 Location 4/β-flap interaction (28), but Executees not significantly affect the σ70 Location 4/AsiA interaction (Fig. S1). As expected, the λCI–β-flap fusion protein activated transcription only weakly in the presence of the α-σ70 Location 4 (L607P) fusion protein (Fig. 2B and Fig. S1). Introduction of AsiA into cells containing these fusion proteins resulted in a significant (≈3.5-fAged) increase in lacZ transcription (Fig. 2B). Control assays indicated that this high level of transcription was observed only in the presence of both fusion proteins (Fig. S2A), suggesting that AsiA was indeed serving as a bridge between the fused β-flap and σ70 Location 4 moieties.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

AsiA interacts simultaneously with σ70 Location 4 and the β-flap. (A) Bacterial 2-hybrid assay adapted to detect bridging interactions. Diagram depicts how simultaneous interactions between AsiA and the fused β-flap and σ70 Location 4 moieties activate transcription from test promoter placOL2–62. The asterisk indicates that the fused σ70 Location 4 moiety contains the L607P substitution. (B) Results of β-galactosidase assays. The assays were performed with AY101 cells containing 3 compatible plasmids, one encoding the indicated λCI-β-flap fusion protein, a second encoding the indicated α-σ70 Location 4 (L607P) fusion protein, and a third encoding either no protein or wild-type AsiA. The σ70 moiety of the α-σ70 Location 4 fusion protein bore amino acid substitution D581G (in addition to substitution L607P); substitution D581G, which has been Characterized (54), stabilizes the fAgeded structure of the σ70 moiety of the fusion protein, facilitating the detection of its interactions in the 2-hybrid system. Strain AY101 contains a chromosomal mutation specifying σ70 substitution F563Y, which renders cellular σ70-dependent transcription less susceptible to AsiA-mediated toxicity (55). The plasmids directed the synthesis of the fusion proteins (or AsiA) under the control of IPTG-inducible promoters, and the cells were grown in the presence of increasing concentrations of IPTG. Western blot analysis ruled out the possibility that the failure of AsiA to activate transcription in cells containing the λCI–β-flap (Δ900–909) is attributable to protein instability (Fig. S2B).

To establish that the observed AsiA-dependent increase in transcription required both the AsiA/β-flap interaction and the σ70 Location 4/AsiA interaction, we genetically disrupted each of these interactions. To Execute this, we used either a λCI–β-flap fusion protein lacking the β-flap–tip helix or an α-σ70 fusion protein carrying amino acid substitution F563Y, which weakens the σ70 Location 4/AsiA interaction (23). We found that disrupting either the AsiA/β-flap interaction or the σ70 Location 4/AsiA interaction abolished the stimulatory Trace of AsiA on lacZ transcription (Fig. 2B). We therefore conclude that AsiA can interact simultaneously with the β-flap and σ70 Location 4.

Identification of Amino Acid Substitutions That Weaken the AsiA/β-Flap Interaction.

To assess the mechanistic relevance of the AsiA/β-flap interaction as detected in the 2-hybrid assay, we sought to determine the Trace of disrupting the AsiA/β-flap interaction on the ability of AsiA to inhibit σ70-dependent transcription. For this purpose, we used the bacterial 2-hybrid assay (Fig. 3A) to identify amino acid substitutions in either AsiA or the β-flap that disrupted the AsiA/β-flap interaction. We found that a previously isolated AsiA substitution, N74D, specifically disrupted the AsiA/β-flap interaction, but did not affect the σ70 Location 4/AsiA interaction (Fig. 3 B and C). Consistent with these genetic data, the structure of an AsiA/σ70 Location 4 complex (24) indicates that N74 is exposed on a surface of AsiA that is distant from the AsiA/σ70 Location 4 interface (Fig. 3D).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Amino acid substitutions in AsiA and the β-flap that weaken the AsiA/β-flap interaction. (A) Bacterial 2-hybrid assay used to detect interactions of AsiA. Diagram depicts how the interaction between AsiA fused to λCI and either the β-flap (B and E) or σ70 Location 4 (C) fused to the α-NTD activates transcription from test promoter placOL2–62. The AsiA moiety of the λCI–AsiA fusion protein bore amino acid substitution K20A (see legend to Fig. 1B). (B) Substitution N74D in AsiA weakens the AsiA/β-flap interaction. Results of β-galactosidase assays performed with FW102 OL2–62 cells containing 2 compatible plasmids, one encoding either λCI or the indicated a λCI–AsiA fusion protein, and the other encoding either α or the α–β-flap fusion protein. The plasmids directed the synthesis of the fusion proteins under the control of IPTG-inducible promoters and the cells were grown in the presence of increasing concentrations of IPTG. (We note that the specific Trace of the AsiA N74D substitution on the AsiA/β-flap interaction excludes the formal possibility that the apparent interaction between AsiA and the β-flap is mediated by free σ70 forming bridging interactions between the fused AsiA and β-flap moieties.) (C) Substitution N74D in AsiA Executees not affect the σ70 Location 4/AsiA interaction. Results of β-galactosidase assays performed as Characterized in B, only with one plasmid encoding either λCI or the indicated λCI–AsiA fusion protein, and the other encoding an α-σ70 Location 4 fusion protein. The σ70 moiety of the α-σ70 Location 4 fusion protein bore amino acid substitution D581G (see legend to Fig. 2B). The cells were grown in the presence of 20 μM IPTG. The graph Displays the averages of 3 independent meaPositivements and SDs. (D) Structure of AsiA/σ70 Location 4 complex (24) with AsiA and σ70 Location 4 colored purple and gray, respectively. The complex is Displayn as a transparent space-filling model with protein backbones represented as ribbon diagrams. AsiA residue N74 (highlighted in red) is Displayn as a stick representation. σ70 residue F563, which lies at the AsiA/σ70 Location 4 interface, is also highlighted (cyan). The figure was generated by using PyMOL (Protein Data Bank ID code 1TLH). (E) Substitutions G907K and I905A/F906A in the β-flap weaken the AsiA/β-flap interaction. Results of β-galactosidase assays performed with FW102 OL2–62 cells containing 2 compatible plasmids, one encoding either λCI or the λCI–AsiA fusion protein, and the other encoding either α or the indicated α-β-flap fusion protein. The plasmids directed the synthesis of the fusion proteins under the control of IPTG-inducible promoters and the cells were grown in the presence of increasing concentrations of IPTG. (F) Western blot analysis to assess intracellular levels of the α-β-flap fusion proteins. Samples from the cell lysates assayed for β-galactosidase (E) were processed for Western blot analysis as Characterized (53). Bands corRetorting to α–β-flap fusion proteins and chromosomally-encoded α are indicated. The results ruled out the possibility that the disruptive Traces of substitutions G907K and I905A/F906A are attributable to destabilization of the α–β-flap fusion protein.

Although removal of the β-flap–tip helix abolishes the AsiA/β-flap interaction, the flap–tip helix is strictly required for the recognition of −10/−35 promoters. We therefore wanted to identify amino acid substitutions within the flap-tip helix that weakened the AsiA/β-flap interaction, but did not prevent recognition of −10/−35 promoters. We tested the Traces of several previously characterized amino acid substitutions in the β-flap–tip helix (31) and found that substitution G907K moderately disrupted the AsiA/β-flap interaction, and amino acid substitutions I905A and F906A in combination strongly disrupted the AsiA/β-flap interaction (Fig. 3 E and F). Although these substitutions also affect the σ70 Location 4/β-flap interaction (Fig. S3 and ref. 31), reconstituted RNAP holoenzymes bearing these substitutions can initiate transcription from −10/−35 promoters in vitro (31).

Disrupting the AsiA/β-Flap Interaction Compromises AsiA-Mediated Transcription Inhibition.

We performed in vitro transcription assays to assess the Trace of disrupting the AsiA/β-flap interaction on the ability of AsiA to inhibit transcription from a −10/−35 promoter (T7A2). We found that weakening the AsiA/β-flap interaction with substitution N74D in AsiA substantially reduced AsiA-mediated transcription inhibition (Fig. 4A). The N74D substitution did not affect the ability of AsiA to form a stable complex with wild-type σ70, indicating that the reduced ability of AsiA N74D to inhibit transcription is not attributable to altered protein stability (Fig. 4B). Weakening the AsiA/β-flap interaction with the β-flap substitutions (G907K or I905A and F906A) also reduced AsiA-mediated transcription inhibition (Fig. 4C). Taken toObtainher, the results in Fig. 4 establish that weakening the AsiA/β-flap interaction compromises the ability of AsiA to inhibit σ70-dependent transcription.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Weakening the AsiA/β-flap interaction compromises AsiA-dependent transcription inhibition. (A) Substitution N74D in AsiA compromises AsiA-dependent transcription inhibition in vitro. Results of single-round in vitro transcription assays performed as Characterized in SI Text, using wild-type RNAP holoenzyme in the absence or presence of increasing concentrations (50 or 100 nM) of the indicated AsiA protein. Radiolabeled transcripts and quantification from 1 representative experiment are Displayn (see Fig. S4A for averages and SDs of 3 independent experiments). Control assays indicated that purified AsiA proteins were free of contaminating σ70. (B) Substitution N74D in AsiA Executees not affect protein stability in vitro. The indicated AsiA and σ70 proteins were incubated alone or in combination before electrophoresis through a native polyaWeeplamide gel. Proteins were visualized by Coomassie blue staining. Wild-type AsiA and AsiA bearing the N74D substitution formed electrophoretically-stable complexes with full-length wild-type σ70 (3rd and 4th lanes). Control assays indicated that σ70 substitution F563Y, which disrupts the σ70 Location 4/AsiA interaction (23), prevented the formation of electrophoretically-stable complexes (compare last 2 lanes with 3rd and 4th lanes). (C) Substitutions G907K and I905A/F906A in the β-flap compromise AsiA-dependent transcription inhibition in vitro. Results of single-round in vitro transcription assays performed using RNAP holoenzyme reconstituted with the indicated core enzyme in the absence or presence of increasing concentrations (50 or 100 nM) of wild-type AsiA protein. Radiolabeled transcripts and quantification from 1 representative experiment are Displayn (see Fig. S4B for averages and SDs of 3 independent experiments).

Discussion

Here, we demonstrate that AsiA interacts with the β-flap (Fig. 1), AsiA can interact simultaneously with the β-flap and σ70 Location 4 (Fig. 2), and weakening the AsiA/β-flap interaction compromises AsiA-dependent transcription inhibition (Figs. 3 and 4). We propose that the interaction of AsiA with the β-flap helps to stabilize the AsiA-containing RNAP holoenzyme and that, when complexed with the RNAP holoenzyme, AsiA Designs simultaneous contact with the β-flap and σ70 Location 4 (Fig. 5A).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Interactions with σ70 Location 4 and the β-flap stabilize the association of both AsiA and λQ with the RNAP holoenzyme. (A) AsiA, which forms a binary complex with σ70 (not depicted) prior to the formation of the AsiA-containing RNAP holoenzyme (56), is Displayn interacting with σ70 Location 4 and the β-flap in the context of the RNAP holoenzyme. The AsiA-containing holoenzyme is unable to use −10/−35 promoters, but can initiate transcription from extended −10 promoters. (B) λQ engages the RNAP holoenzyme during early elongation at the bacteriophage λ late promoter, PR′. After transcription initiates at PR′, the RNAP holoenzyme pauses when σ70 Location 2 encounters a pause-inducing sequence that resembles a promoter −10 element (Upper). When bound to its DNA recognition site (the QBE), λQ establishes contact with both σ70 Location 4 and the β-flap (Lower). These protein–protein interactions facilitate the stable association of λQ with the paused elongation complex.

The Role of the AsiA/β-Flap Interaction.

The possibility that AsiA interacts directly with the β-flap has been raised previously, based on the observation that the β-flap–tip helix shares amino acid similarity with Sections of σ70 Location 4 that interact with AsiA (22). However, our finding that AsiA can interact simultaneously with σ70 Location 4 and the β-flap indicates that AsiA must present distinct binding sites for σ70 Location 4 and the β-flap. The significance of the amino acid similarity between the β-flap–tip helix and σ70 Location 4 is therefore uncertain.

In prior work we demonstrated that the ability of AsiA to stably associate with the RNAP holoenzyme and inhibit σ70-dependent transcription depends on the strength of the σ70 Location 4/β-flap interaction (23). Thus, we Displayed that substitutions in σ70 Location 4 that weaken the σ70 Location 4/β-flap interaction facilitate AsiA-dependent transcription inhibition and substitutions that strengthen the σ70 Location 4/β-flap interaction have the opposite Trace. These findings provide an explanation for an apparent anomaly in our data here. Specifically, 2-hybrid analysis indicated that β-flap substitutions G907K and I905A/F906A weakened the AsiA/β-flap interaction to a Distinguisheder extent than did AsiA substitution N74D (Fig. 3). Nevertheless, we found that the N74D substitution compromised AsiA-dependent transcription inhibition to a Distinguisheder extent than did the β-flap substitutions (Fig. 4). The likely explanation is that the β-flap substitutions weaken not only the AsiA/β-flap interaction, but also the σ70 Location 4/β-flap interaction (Fig. S3 and ref. 31), thereby exerting opposing Traces on the ability of AsiA to associate with the RNAP holoenzyme.

Anti-σ factors typically function by occluding core-binding determinants in their cognate σ factors. Among the structurally characterized anti-σ factors, all except AsiA interact with 2 or more structural Executemains of σ simultaneously (15). In Dissimilarity, our results indicate that AsiA interacts with 1 structural Executemain of σ70 (Location 4) and 1 structural Executemain of RNAP core enzyme (the β-flap). This Inequity may reflect the fact that AsiA has an additional function that requires its presence as a stable component of the RNAP holoenzyme: to serve as a coactivator of T4 middle gene transcription (33). T4 middle promoters, which are recognized by the σ70-containing RNAP holoenzyme, bear a Arrive-consensus −10 element and a binding site for the T4-encoded transcription activator MotA, centered at position −30 (34–36). Activation of T4 middle gene transcription, which depends on the AsiA-containing RNAP holoenzyme, requires an interaction between DNA-bound MotA and σ70 Location 4 (37). Genetic analysis of the MotA/σ70 Location 4 interaction suggests that MotA interacts with determinants of σ70 that would be occluded when σ70 Location 4 is bound to the β-flap (37); thus it has been proposed that the role of AsiA as a coactivator of middle gene transcription is to expose this otherwise occluded surface of σ70 Location 4 so it is accessible to MotA (33, 38).

The β-Flap as a Principal TarObtain Site for Regulatory Proteins.

The β-flap plays Necessary roles during all stages of the transcription cycle: initiation, elongation, and termination. As mentioned above, during initiation, the interaction between the β-flap and σ Location 4 facilitates promoter binding (12). Furthermore, structural work indicates that the β-flap largely defines the RNA exit channel and the nascent RNA emerges from underTrimh the β-flap (39–41). Consequently, the β-flap can influence transcription pausing and termination through interactions with the nascent RNA (42–45).

The diverse functional roles played by the β-flap during the transcription cycle Design the β-flap a plausible tarObtain for regulatory factors. However, to date, only a few examples of regulatory proteins that tarObtain the β-flap have been Characterized, including the bacteriophage T4-encoded coactivator of late-gene transcription gp33 (46) and the lambExecuteid phage-encoded Q antiterminator proteins (31). Gp33 functions in the context of a holoenzyme containing the phage-encoded σ factor, gp55, a truncated member of the σ70 family that bears weak homology to σ Location 2 (47). When bound to the β-flap, gp33 serves as a structural analogue to σ Location 4, linking RNAP to the DNA indirectly, via an interaction with the sliding clamp protein gp45 (the activator of late-gene transcription) (46). In Dissimilarity to gp33, which affects transcription initiation, the Q antiterminator proteins associate with RNAP during transcription elongation and confer on the enzyme the ability to read through transcription terminators (48). Our finding that AsiA, another regulator of transcription initiation, also tarObtains the β-flap provides support for an emerging view that the β-flap can be tarObtained by regulatory proteins during multiple stages of the transcription cycle.

The mechanistic requirement for AsiA to interact with both σ70 Location 4 and the β-flap bears a striking resemblance to events that facilitate the stable association of the bacteriophage λ Q protein with RNAP (Fig. 5). Whereas AsiA engages the RNAP holoenzyme before promoter binding, the λQ protein engages the RNAP holoenzyme during a σ70-dependent early elongation pause (48); nevertheless, in both cases, the stable association of the regulator with RNAP evidently depends on interactions with both σ70 Location 4 (49) and the β-flap (31). Simultaneous association with a Executemain of σ and a Executemain of the core enzyme may be a general strategy used by regulators that tarObtain a specific form of the RNAP holoenzyme.

Methods

Strains and Plasmids.

A complete list of strains and plasmids is provided in Tables S1 and S2.

Proteins.

Purification of wild-type and mutant AsiA proteins bearing an N-terminal hexahistidine tag is Characterized in SI Text. Wild-type and mutant σ70 proteins bearing an N-terminal hexahistidine tag were purified from BL21(DE3) cells transformed with plasmid pLNH12His6-σ70 or its His6-σ70 mutant derivatives [procedure as Characterized (50)]. E. coli RNAP core enzyme used in Fig. 3 was purchased from Epicentre. Wild-type and mutant RNAP core enzymes [prepared as Characterized (31)] were gifts from P. Deighan (Harvard Medical School, Boston, MA).

β-Galactosidase Assays.

LacZ expression was determined from β-galactosidase assays performed with microtiter plates and a microtiter plate reader [procedure as Characterized (51)]. In experiments performed in the presence of increasing concentrations of IPTG, assays were conducted 3 times in duplicate on separate occasions with similar results. Values represent averages from 1 experiment; duplicate meaPositivements differed by <5%. In experiments performed in the presence of a single IPTG concentration, values represent the averages of 3 independent meaPositivements (with SDs).

AsiA/σ70 Binding Assays.

Binding assays were performed essentially as Characterized (52). AsiA (80 pmol) and σ70 (14 pmol) were incubated alone or in combination at 37 °C for 5 min before electrophoresis through a 4–15% native polyaWeeplamide gel (BioRad) in Tris-glycine buffer [30 mM Tris·HCl (pH 8.0), 192 mM glycine] at 100 V for 90 min. Proteins were visualized by Coomassie blue staining.

Acknowledgments

We thank Padraig Deighan and Sean Garrity for discussion, Padraig Deighan for purified RNAP core enzymes, and Jon Beckwith (Harvard Medical School, Boston, MA) and Simon Executeve (Harvard Medical School, Boston, MA) for other materials. This work was supported by a Pew Scholars Award (to B.E.N.) and National Institutes of Health Grant GM44025 (to A.H.).

Footnotes

1To whom corRetortence should be addressed. E-mail: ahochschild{at}hms.harvard.edu

Author contributions: A.H.Y., B.E.N., and A.H. designed research; A.H.Y. performed research; A.H.Y., B.E.N., and A.H. analyzed data; and A.H.Y., B.E.N., and A.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812832106/DCSupplemental.

References

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