Abstract
RNA polymerase (RNAP) melts promoter DNA to form transcription-competent open promoter complex (RPo). Interaction of the RNAP σ subunit with non-template strand bases of a conserved −10 element (consensus sequence T−12A−11T−10A−9A−8T−7) is an important source of energy-driving localized promoter melting. Here, we used an RNAP molecular beacon assay to investigate interdependencies of RNAP interactions with −10 element nucleotides. The results reveal a strong cooperation between RNAP interactions with individual −10 element non-template strand nucleotides and indicate that recognition of the −10 element bases occurs only when free energy of the overall RNAP −10 element binding reaches a certain threshold level. The threshold-like mode of the −10 element recognition may be related to the energetic cost of attaining a conformation of the −10 element that is recognizable by RNAP. The RNAP interaction with T/A−12 base pair was found to be strongly stimulated by RNAP interactions with other −10 element bases and with promoter spacer between the −10 and −35 promoter elements. The data also indicate that unmelted −10 promoter element can impair RNAP interactions with promoter DNA upstream of the −11 position. We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of RPo formation.
Highlights
Formation of the transcription-competent open promoter complex (RPo) by bacterial DNA-dependent RNA polymerase (RNAP) is a critical checkpoint on the pathway of gene expression
We suggest that the threshold effect contributes to the selectivity of open promoter complex formation by hindering RNAP binding to non-promoter DNA
We measured how changing the identity of one À10 element nucleotide affects RNAP affinity to other À10 element nt-strand nucleotides in the context of promoter fragment DNA probes
Summary
Formation of the transcription-competent open promoter complex (RPo) by bacterial DNA-dependent RNA polymerase (RNAP) is a critical checkpoint on the pathway of gene expression. Oligonucleotides and fork junction promoter fragments containing single-stranded (ss) extensions corresponding to the nt-strand of the À10 promoter element have been used as model substrates to study RNAP interactions with melted DNA in promoter complexes [3,4]. Binding studies using these DNA probes confirmed that in the context of the RNAP holoenzyme, s subunit recognizes the À10-ntstrand sequence in the single-stranded form [3,4,5,6,9,17].
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