Function-Based Selection and Characterization of Base-Pair Polymorphisms in a Promoter of Escherichia coli RNA Polymerase-ς 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-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.Gene expression is often regulated at transcription initiation. In the absence of gene regulatory proteins, the rate of initiation at a particular promoter depends on the concentration of RNA polymerase and the sequence of the promoter element. In both prokaryotes and eukaryotes, transcription initiation is a multistep process that begins with sequencespecific recognition and proceeds through formation of two or more intermediate RNA polymerase-promoter complexes prior to synthesis of the first phosphodiester bond of the product RNA (31). Subsequently, the initiated complex isomerizes into one that generates full-length RNA products (for exceptions, see references 18 and 34). The fractional occupancy of a promoter by RNA polymerase and the rate at which RNA polymerase proceeds through the initiation pathway determine, in part, the amount of RNA produced from a gene.The pathway for transcription initiation is best understood in prokaryotes (Fig. 1A). In these organisms, RNA polymerase binds to promoter DNA and initially forms an unstable "closed" complex that dissociates with the addition of polyanionic competitors such as single-stranded DNA or heparin (31). The closed complex isomerizes through a number of intermediate complexes (21,33) in which the DNA at the transcription start site remains base paired (Fig. 1A). Subsequently, these intermediates isomerize to form one or more competitor-resistant "open" complexes, in which the DNA strands surrounding the transcription start site display increasing separation (4, 36). RNA polymerase initiation complexes form after the DNA in the region between Ϫ11 and ca. ϩ5 is fully denatured and the synthesis of small (8-to 12-base) "abortive" oligonucleotide products takes place and/or the polymerase escapes from the promoter, forming an elongation complex competent to synthesize full-length RNA transcripts.In prokaryotes, both the affinity of RNA polymerase for DNA and the rate of isomerization of the RNA polymerase-DNA complexes to form the various intermediate species dep...

The transition from transcriptional initiation to elongation

“…In E. coli, such differences can affect both the affinity of RNAP for the promoter and the rate of post-recruitment steps [37], such as the level of abortive initiation and promoter escape [38,39], the formation of unproductive "moribund" RNAP complexes [40,41], and σ-dependent pausing of RNAP in the initial transcribed region [42,43]. As initial transcription occurs through scrunching, an intriguing possibility is that the presence of promoter-proximal σ-dependent pause sites may cause RNAP to favor forward translocation and hence promoter escape, rather than repeated cycles of abortive initiation.…”

Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters

“…Previous reports have described optimization by in vitro selection of double-stranded naturally occurring promoters for phage and bacterial RNA polymerases (24,25). The present experiments are distinct from this by involving only singlestranded sequence and using no naturally occurring sequence as the starting point.…”

Molecular Manipulations for Enhancing Luminescent Bioreporters Performance in the Detection of Toxic Chemicals

“…Indeed, a much faster and stronger induction, accompanied by an improvement of detection sensitivity, was achieved. In this regard, it is worth noting that when the -10 consensus sequence was changed into a combination that took into account the multiple pathway of transcription initiation [18], induction by SOS-activating agents was completely abolished.…”