The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma70 subunit of Escherichia coli RNA polymerase

Abstract

Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the sigma70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with sigma70. We show here that the N-terminal half of MotA (MotA(NTD)), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of sigma70 with comparable sigma38 residues abolishes the interaction with MotA(NTD) in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA(CTD)) binds DNA with a K(D(app)) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of sigma70 helps to substitute functionally for an interaction between sigma70 and a promoter -35 element.

FIG. 1.

Formation of discrete complexes of AsiA-σ⁷⁰ and MotA-σ⁷⁰ requires the last 43 amino acids of σ⁷⁰. A 7.25-μl mixture containing 17 mM Tris-Cl (pH 7.9), 280 mM NaCl, 22% glycerol, 0.7 mM EDTA, 0.7 mM 2-mercaptoethanol, 0.04 mM dithiothreitol, and (as indicated by + or −) 80 pmol of AsiA, 50 pmol of MotA, 14 pmol of σ⁷⁰, and 14 pmol of σ^1-570 was incubated at 37°C for 5 min. Samples were then subjected to electrophoresis in a 6% polyacrylamide native protein gel, and proteins were detected after staining with colloidal Coomassie blue (Invitrogen). The positions of free σ⁷⁰ and free σ^1-570 are indicated. (Both the σ⁷⁰ and the σ^1-570 preparations contain a slow-moving band that is consistent with the presence of σ dimer.) The locations of the AsiA-σ⁷⁰ complex, the MotA-σ⁷⁰ complex, and the AsiA-σ⁷⁰-MotA complex are indicated by arrowheads.

FIG. 2.

E. coli two-hybrid system for detecting interactions between the C-terminal region of σ⁷⁰ and other proteins. The cartoon depicts the positions of RNA polymerase subunits β, β′, and σ⁷⁰ and the α-σ⁷⁰ chimera, which consists of the N-terminal domain of α fused to the C terminus of σ⁷⁰, at a promoter upstream of the reporter lacZ gene. The cI-bait fusion protein is located at position −62. (See text for details.)

FIG. 3.

AsiA, MotA, and MotA^NTD each interact with the C-terminal region of σ⁷⁰ in the E. coli two-hybrid assay. β-Galactosidase (β-gal) activity is plotted versus IPTG concentration for KS1 cells containing pBRα-σ⁷⁰ and pcI-AsiA (•), pcI-MotA^NTD (▪), pcI-MotA^fl (▴), pcI-Mot21^NTD (□), pcI-MotA⁻ (▵), or pACλcI32 (○). Cultures were grown continuously in the presence of the indicated concentrations of IPTG. Points and standard deviations (indicated by error bars) represent the averages of the results of three assays. In this assay, in which cultures were grown continuously in the presence of IPTG, MotA^NTD gave higher levels of β-galactosidase activity than did MotA^fl. However, in assays in which cultures were grown for only 1 h with IPTG, the activities seen with MotA^NTD and MotA^fl were similar (data not shown).

FIG. 4.

MotA^NTD does not interact with the C-terminal region of σ³⁸. β-Galactosidase (β-gal) activity is plotted versus IPTG concentration for KS1 cells containing pBRα-σ³⁸ and pcI-AsiA (•), pcI-MotA^NTD (▪), pcI-MotA^fl (▴), pcI-Mot21^NTD (□), or pACλcI32 (○). Cultures were grown to mid-log phase in the absence of IPTG and then grown in the presence of the indicated IPTG concentrations for 1 h. Points and standard deviations (indicated by error bars) represent the averages of the results of two assays.

FIG. 5.

The MotA-σ⁷⁰ interaction in the E. coli two-hybrid assay requires the last 17 amino acids of σ⁷⁰. Relative β-galactosidase activity is shown for assays with cI-AsiA (top panel) or cI-MotA^NTD (bottom panel) with α-σ⁷⁰/σ³⁸, α-σ⁷⁰(R596H), α-σ⁷⁰(H600A), α-σ⁷⁰(H600R), or α-σ⁷⁰(R608C). (See Materials and Methods for the determination of relative β-galactosidase activity.) Points and standard deviations (indicated by error bars) represent the averages of the results of two to eight assays.

FIG. 6.

Polymerase reconstituted with σ^Δ608-613 or σ^Δ604-613 is defective for MotA-dependent transcription at P_uvsX. AsiA (23 pmol) and the indicated σ⁷⁰ (1.3 pmol) were incubated for 10 min at 37°C in 2.5 μl of incubation buffer I. The mixture was placed on ice, and then 0.5 pmol of core polymerase in 2.89 μl of incubation buffer II was added. Transcription was initiated by adding an aliquot (2.16 μl) of the resulting solution to 2.35 μl of DNA buffer containing 0.02 pmol of linearized pDKT90 DNA with or without 1.9 pmol of MotA. (A) The ³²P-labeled P_uvsX RNA obtained after a set of single-round transcription reactions; (B) quantitation of the results from three independent reactions. For each σ⁷⁰, the values shown for +AsiA, +MotA, and +AsiA/MotA were normalized relative to a value of 1 for that polymerase alone.

FIG. 7.

A C-terminal peptide of MotA binds DNA. Gel retardation assays, which contained 0.5 pmol of the ³²P-labeled 74-bp P_uvsX DNA, 26 ng of poly(dI-dC) competitor DNA, and buffer (lane 1), protein fraction from uninduced BL21(DE3) cells containing the plasmid with MotA^{cloned CTD} (lane 2), or 16 pmol of MotA^{cloned CTD} protein in a purified fraction from induced BL21(DE3) cells containing the plasmid with MotA^{cloned CTD} (lane 3), are shown. The fraction used in lane 2 was purified in a manner similar to that of the fraction used in lane 3.

FIG. 8.

Model of MotA/AsiA activation at a T4 middle promoter. The cartoon depicts the positions of σ⁷⁰, AsiA, and MotA at a T4 middle promoter. MotA^CTD interacts with the MotA box motif (5′ [T/A][T/A]TGCTT[T/C]A 3′) centered at −30. Both AsiA and MotA^NTD interact with the C-terminal region of σ⁷⁰. The positions of σ⁷⁰ regions 2.4 and 4.2 are shown. (See text for details.)