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 a fluorometric RNAP molecular beacon assay to investigate interdependencies of RNAP interactions with −10 element nucleotides. The data 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 the overall RNAP −10 element binding acquires a free energy below a ca. −3 kcal/mol threshold. These results may be explained by that the individual interactions between RNAP and −10 element nucleotides cooperatively contribute into retaining a characteristic recognizable conformation of the −10 element nt-strand that was revealed in recent structural studies. 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 transcription initiation complex formation.
Published Version
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