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

Abstract

To initiate transcription from specific promoters, the bacterial RNA polymerase (RNAP) core enzyme must associate with the initiation factor sigma, which contains determinants that allow sequence-specific interactions with promoter DNA. Most bacteria contain several sigma factors, each of which directs recognition of a distinct set of promoters. A large and diverse family of proteins known as "anti-sigma factors" regulates promoter utilization by targeting specific sigma factors. The founding member of this family is the AsiA protein of bacteriophage T4. AsiA specifically targets the primary sigma factor in Escherichia coli, sigma(70), and inhibits transcription from the major class of sigma(70)-dependent promoters. AsiA-dependent transcription inhibition has been attributed to a well-documented interaction between AsiA and conserved region 4 of sigma(70). 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 domain of the RNAP beta-subunit (the beta-flap). Our findings support the emerging view that the beta-flap is a target site for regulatory proteins that affect RNAP function during all stages of the transcription cycle.

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 domain (α-NTD), activates transcription from test promoter placO_L2–62, which bears the λ operator O_L2 centered 62 bp upstream of the lac core promoter start site. In reporter strain FW102 O_L2–62, test promoter placO_L2–62 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 O_L2–62 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 described (53). Bands corresponding 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.

Fig. 2.

AsiA interacts simultaneously with σ⁷⁰ region 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 region 4 moieties activate transcription from test promoter placO_L2–62. The asterisk indicates that the fused σ⁷⁰ region 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 region 4 (L607P) fusion protein, and a third encoding either no protein or wild-type AsiA. The σ⁷⁰ moiety of the α-σ⁷⁰ region 4 fusion protein bore amino acid substitution D581G (in addition to substitution L607P); substitution D581G, which has been described (54), stabilizes the folded 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).

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 region 4 (C) fused to the α-NTD activates transcription from test promoter placO_L2–62. 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 O_L2–62 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 effect 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 does not affect the σ⁷⁰ region 4/AsiA interaction. Results of β-galactosidase assays performed as described in B, only with one plasmid encoding either λCI or the indicated λCI–AsiA fusion protein, and the other encoding an α-σ⁷⁰ region 4 fusion protein. The σ⁷⁰ moiety of the α-σ⁷⁰ region 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 shows the averages of 3 independent measurements and SDs. (D) Structure of AsiA/σ⁷⁰ region 4 complex (24) with AsiA and σ⁷⁰ region 4 colored purple and gray, respectively. The complex is shown as a transparent space-filling model with protein backbones represented as ribbon diagrams. AsiA residue N74 (highlighted in red) is shown as a stick representation. σ⁷⁰ residue F563, which lies at the AsiA/σ⁷⁰ region 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 O_L2–62 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 described (53). Bands corresponding to α–β-flap fusion proteins and chromosomally-encoded α are indicated. The results ruled out the possibility that the disruptive effects of substitutions G907K and I905A/F906A are attributable to destabilization of the α–β-flap fusion protein.

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 described 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 shown (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 does not affect protein stability in vitro. The indicated AsiA and σ⁷⁰ proteins were incubated alone or in combination before electrophoresis through a native polyacrylamide 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 region 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 shown (see Fig. S4B for averages and SDs of 3 independent experiments).

Fig. 5.

Interactions with σ⁷⁰ region 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 shown interacting with σ⁷⁰ region 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, P_R′. After transcription initiates at P_R′, the RNAP holoenzyme pauses when σ⁷⁰ region 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 region 4 and the β-flap (Lower). These protein–protein interactions facilitate the stable association of λQ with the paused elongation complex.