Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70)

Abstract

We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma(70) forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus -10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.

FIG. 1

Transcription pathway and sequence of the starting promoter. (A) Proposed transcription initiation pathway for E. coli RNA polymerase, based on evidence described in the text and reference . (B) Sequence of the modified bacteriophage 434 P_R promoter that is the starting material for the selection experiments. The −35 and −10 regions in this promoter are underlined (2, 37).

FIG. 2

The −10 sequences preferred by ς⁷⁰ RNA polymerase for formation of heparin-resistant complexes. Sequences were selected and determined as described in Materials and Methods. Shown is the frequency distribution of the occurrence of a selected base sequence at each position in the selected promoter, as selected on the basis of high-affinity RNA polymerase binding (A) or rapid open-complex formation (B). The percent occurrence of a base pair at each position was derived from analysis of 14 and 22 cloned sequences for panels A and B, respectively.

FIG. 3

Runoff transcription directed by the rate- and affinity-selected promoters. Concentration-dependent (A and B) and time-dependent (C and D) runoff transcription directed by the rate- and affinity-selected promoters was determined in single-round runoff transcription assays, performed as described in Materials and Methods. The products of a typical transcription reaction are shown (A and C) as visualized by a PhosphorImager. Data from three experiments were quantified (B and D). Error bars, standard deviations. The amount of transcript is expressed in PhosphorImager units per microgram of input DNA (p.u.). Symbols in panels B and D represent promoters containing the −10 sequences.

FIG. 4

KMnO₄ reactivity of selected promoters in the presence of saturating RNA polymerase concentrations. Labeled DNA fragments were incubated with RNA polymerase containing the indicated promoters and treated as described in Materials and Methods. The positions of the reactive thymines present on the template strand in the −10 region in all the selected promoters are indicated by the bracketed numbers. The relative KMnO₄ reactivity of the −10 region is indicated below the gel and was determined as described in Materials and Methods.