Environmental regulation operating at the promoter clearance step of bacterial transcription

Abstract

In vivo transcription of the Escherichia coli argO gene, which encodes an arginine (Arg) exporter, requires the LysR-family regulator protein ArgP (previously called IciA) and is induced in the presence of Arg or its naturally occurring antimetabolite analog canavanine. Lysine (Lys) addition, on the other hand, phenocopies an argP mutation to result in the shutoff of argO expression. We now report that the ArgP dimer by itself is able to bind the argO promoter-operator region to form a binary complex, but that the formation of a ternary complex with RNA polymerase is greatly stimulated only in presence of a coeffector. Both Arg and Lys were proficient as coeffectors for ArgP-mediated recruitment of RNA polymerase to, and open complex formation at, the argO promoter, although only Arg (but not Lys) was competent to activate transcription. The two coeffectors competed for binding to ArgP, and the ternary complex that had been assembled on the argO template in the presence of Lys could be chased into a transcriptionally active state upon Arg addition. Our results support a novel mechanism of argO regulation in which Lys-bound ArgP reversibly restrains RNA polymerase at the promoter, at a step (following open complex formation) that precedes, and is common to, both abortive and productive transcription. This represents, therefore, the first example of an environmental signal regulating the final step of promoter clearance by RNA polymerase in bacterial transcription. We propose that, in E. coli cells, the ternary complex remains assembled and poised at the argO promoter at all times to respond, positively or negatively, to instantaneous changes in the ratio of intracellular Arg to Lys concentrations.

Figure 1.

ArgP-mediated regulation by Arg, Lys, and CAN of argO transcription. (A) Sequence of the argO regulatory region used in this study, numbered with respect to the A residue (in bold) representing the start site of transcription (taken as +1). Promoter −35 and −10 motifs and GTG triplet representing start site of translation have lines above them. Inverted-repeat sequence is marked by overhead arrows. The ArgP region from around −85 to −20 (as determined in this study) is underlined and bracketed. The two −10-like motifs in the ITS are italicized. (B) In vivo argO-lac expression, measured as β-galactosidase-specific activities (sp act), in host strain derivatives with plasmid constructs carrying the argO regulatory region, either wild-type (wt) or with different mutations as described in the text. The host strain for top panel data was argP⁺ (except for bar at extreme left, which was from host with argP-null mutation) and that for bottom panel data was argP^d-S94L. (C) Radiolabeled in vitro transcription products obtained with the 427-bp argO template (with sequence from −293 to +109), in reaction mixes with various additives as indicated, and subjected to electrophoresis on denaturing 10% polyacrylamide gel. (RO) Run-off transcript from argO promoter; (EE) end-to-end transcription product; (His) histidine.

Figure 2.

Binary interaction of ArgP with argO regulatory region. In A–C, bands corresponding to free DNA (F) and to DNA in binary complex with ArgP (B) are marked. (A) Electrophoretic mobility of radiolabeled 427-bp argO fragment with indicated monomer concentrations of ArgP in the absence or presence of coeffectors Arg or Lys. (B) Electrophoretic mobility of radiolabeled 427-bp argO fragment (wild type) and its deletion derivatives (described in text) in the absence or presence of 20 nM ArgP (monomer). (C,D) Electrophoretic mobility of radiolabeled fragments corresponding to indicated regions of argO in the absence or presence of ArgP (20 nM monomer) and Lys. Below each lane is given the value, obtained from densitometry, for the fraction of labeled DNA bound by the protein.

Figure 3.

Determination of the ArgP-binding site on argO. DNase I digestion patterns were determined for radiolabeled argO fragments that had been uniquely 5′-end-labeled (as described in Materials and Methods) on either the top strand at −115 (A) or the bottom strand toward +109 (B), in the absence or presence of ArgP and coeffectors Arg (A) or Lys (L). The corresponding di-deoxy sequence ladder of argO is represented alongside the numbered lanes in each of the two panels. Vertical lines denote the regions of ArgP footprint, and nucleotide positions of interest are also marked. (C) Comparison of electrophoretic mobility of argO fragment from −85 to −20 with that of the 427-bp fragment (from −293 to +109), in the absence or presence of ArgP (20 nM monomer) and Lys. (F) Free DNA; (B) ArgP–DNA binary complex. Below each lane is given the value, obtained by densitometry, for the fraction of labeled DNA bound by the protein.

Figure 4.

Ternary interaction of ArgP and RNAP with argO regulatory region. In A–C, bands corresponding to free DNA (F), DNA in binary complex with ArgP [B(ArgP)] or RNAP [B(RNAP)], and that in ternary complex with ArgP and RNAP (T), are marked. (A) Electrophoretic mobility of radiolabeled argO fragment in the absence or presence (as indicated) of 20 nM ArgP (monomer), 80 nM RNAP, and coeffectors Arg, Lys, or CAN at 0.1 mM. (B) Electrophoretic mobility experiment as in A, in the absence of or following treatment with heparin at 200 μg/mL for the time periods indicated in parentheses. (Note that the samples of free DNA probe preparations used in lane 1 of A and lane 2 of B appear to have been inadvertently contaminated with a small amount of ArgP protein.) (C) Electrophoretic mobility experiment as in B, in the presence of 0.5 mM NTPs in all reactions. (D) In vivo KMnO₄ footprinting in cultures of argP mutant (argP⁻) and argP⁺ strain grown in 0.2% glucose–minimal A medium without (Nil) or with 1 mM Arg or Lys supplementation as indicated, and then treated with rifampicin. The corresponding di-deoxy sequence ladder of argO is represented alongside, and the −10 region of the argO promoter as well as the HaeIII truncation site at −80 are marked.

Figure 5.

Absence of short products representing paused or abortive transcripts in argO transcription reactions with ArgP and Lys. In vitro transcription products (abortive, run-off from argO promoter [RO], and end-to-end [EE], as marked) from the 427-bp argO template (with sequence from −293 to +109) in reaction mixes with various additives as indicated were subjected to electrophoresis on denaturing 20% polyacrylamide gel.

Figure 6.

Exonuclease III (Exo III) protection assays to determine upstream and downstream edges of protection on argO by ArgP and RNAP. argO fragments that had been uniquely 5′-end-labeled (as described in Materials and Methods) on either the bottom strand toward +109 (A) or the top strand at −115 (B) were used in reaction mixes with various additives as indicated. (A) Arg; (L) Lys. The corresponding di-deoxy sequence ladder of argO is represented alongside the numbered lanes in each of the two panels. Arrows denote protected bands at the indicated nucleotide positions in the two panels. Lanes 7 and 8 in B represent the reactions involving chase with Arg (0.1 mM and 10 mM, respectively), as described in the text.

Figure 7.

Arg (or CAN) chase of ArgP–RNAP–argO complex assembled in the presence of Lys and NTPs, either in solution (A) or on streptavidin-coated beads (B). See text for details. Unless otherwise indicated, ArgP was added to all reaction mixes. Other additions were as indicated above the lanes: The primary additives were those that were present in the initial reactions, and the secondary additives were those that were added during the chase step. (A) Arg; (L) Lys; (C) CAN; (RO) run-off transcript from argO promoter; (EE) end-to-end transcription product. In B, the fractions from the primary reaction mixes (pellet [P]; supernatant [S]; unseparated primary reaction mix [P + S]) that were used for preparation of samples for loading on the corresponding lanes of the gel are indicated. Transcription products were resolved on denaturing 6% (A) or 10% (B) polyacrylamide gels.

Figure 8.

Binding studies of coeffectors to Arg. Indicated in each chromatograph panel is the material (protein, labeled amino acid, and/or cold competitor amino acid in 100-fold excess) that was loaded on the column. See the text for details. A represents the plot of fluorescence intensity (in arbitrary units, AU) in the various fractions, and B–I represent plots of radioactivity measurements (in counts per min, cpm) in the fractions. (Ala) Alanine.