Supercoil-induced extrusion of a regulatory DNA hairpin

Abstract

Bacteriophage N4 virion RNA polymerase (N4 vRNAP) promoters contain inverted repeats, which form a 5- to 7-base-pair stem, 3-base loop hairpin that is required for vRNAP recognition. We show that, contrary to certain theoretical predictions, hairpin extrusion can occur at physiological superhelical densities in a Mg2+-dependent manner. Specific sequences on the template strand are required for hairpin extrusion. These sequences define stable DNA hairpins that are relatively unreactive to single strand-specific probes. In addition, a specific stable hairpin-inducing sequence can regulate transcription in vivo. Thus, a DNA structure, in its natural environment, is involved in transcriptional regulation.

Figure 1

Dependence on superhelical density of the reactivity of promoter P2 nontemplate (Upper) and template (Lower) strands to different probes: CAA (Left), MBN (Center), and T7 endo I (Right). The sequences of the two strands (arrows indicate inverted repeats) are shown next to the gels. Short arrows, modification or cleavage sites; σ̄ is superhelical density.

Figure 2

Two-dimensional gel electrophoresis of N4 promoter-containing circles. Topoisomers of a 2.2-kb circle containing a single N4 promoter were subjected to two-dimensional gel electrophoresis before (Upper) or after (Lower) treatment with T7 endo I. The arrowhead marks the topoisomer with a linking number of 7, r indicates the relaxed topoisomer, and l indicates linear DNA.

Figure 3

Effect of cations on hairpin extrusion at promoter P2. (A) Effect of cations on CAA reactivity of promoter P2 nontemplate strand. Reactions with CAA were performed in the absence and presence of cations as indicated at the top of the lanes. (B) Effect of complex cations on MBN reactivity of promoter P2 nontemplate strand. The short arrows indicate the sites at which Vent DNA polymerase terminates as a result of modification or cleavage.

Figure 4

Reactivity of wild-type promoter P2 and mutant promoter P2flip to different probes. At the top, promoter sequences; mutated bases in P2flip are shown in boldface. Triangles and small arrows indicate sites of T7 endo I and CAA modification, respectively. Upper pair of gels, CAA; lower pair, T7 endo I.

Figure 5

Promoter sequences required for hairpin extrusion. The sequence of the wild-type promoter template strand is shown. X and X′ can be any nucleotide as long as they can base pair. Mutations tested for hairpin extrusion are shown indicating the presence (+) or absence (−) of extrusion from circular templates of physiological superhelical density.

Figure 6

In vivo hairpin extrusion. (A) Primer extension analysis of RNAs synthesized in E. coli cells from promoters P1 or P1 G-13 and the respective P2 control promoters. P1 and P1 G-13 promoter template-strand sequences are shown. Short arrow, primer extension product. (B) Primer extension analysis of RNAs synthesized in E. coli cells from rrnB P1 wild-type or mutant promoters containing extruding and nonextruding hairpin sequences between the −10 and −35 hexamers.