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Cooperativity and interaction energy threshold effects in recognition of the -10 promoter element by bacterial RNA polymerase.

Mekler V, Severinov K - Nucleic Acids Res. (2013)

Bottom Line: 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 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.We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of RPo formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Waksman Institute of Microbiology Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA and Institutes of Molecular Genetics and Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.

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.

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Effect of the T−12A substitution on RNAP binding to fork junction probes. Sequence of double-stranded parts of fork junction probes 15–18 is shown on the top of the figure.
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gkt541-F5: Effect of the T−12A substitution on RNAP binding to fork junction probes. Sequence of double-stranded parts of fork junction probes 15–18 is shown on the top of the figure.

Mentions: A T at the −12 position is highly conserved among bacterial σ70-dependent promoters (23) and substitutions of −12T decrease transcription from many promoters (24,25). Substitution of a T/A−12 base pair for an A/T base pair considerably decreases heparin resistance of RNAP complexes with fork junctions based on the lacUV5 promoter (32). In agreement with these data, we found that T−12A substitution decreased affinities of fork junction probes 5, 6(−11T) and 9 by 170- to 260-fold (Figure 5). Heparin resistance assay data and structural modeling indicate that both nt-strand T and t-strand A of the T/A−12 base pair are recognized by the σ subunit (4,18). Consistently, we found that affinity of fork junction probe 8 bearing an unpaired T at −12 was less affected by the T-12A substitution than affinities of probes 5, 6(−11T) and 9 (Figure 5). In agreement with this result, a derivative of fork junction probe 5 lacking the template strand nucleotide at position −12 (probe 31) bound RNAP ∼10-fold weaker than probe 5 (Supplementary Figure S3).Figure 5.


Cooperativity and interaction energy threshold effects in recognition of the -10 promoter element by bacterial RNA polymerase.

Mekler V, Severinov K - Nucleic Acids Res. (2013)

Effect of the T−12A substitution on RNAP binding to fork junction probes. Sequence of double-stranded parts of fork junction probes 15–18 is shown on the top of the figure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753650&req=5

gkt541-F5: Effect of the T−12A substitution on RNAP binding to fork junction probes. Sequence of double-stranded parts of fork junction probes 15–18 is shown on the top of the figure.
Mentions: A T at the −12 position is highly conserved among bacterial σ70-dependent promoters (23) and substitutions of −12T decrease transcription from many promoters (24,25). Substitution of a T/A−12 base pair for an A/T base pair considerably decreases heparin resistance of RNAP complexes with fork junctions based on the lacUV5 promoter (32). In agreement with these data, we found that T−12A substitution decreased affinities of fork junction probes 5, 6(−11T) and 9 by 170- to 260-fold (Figure 5). Heparin resistance assay data and structural modeling indicate that both nt-strand T and t-strand A of the T/A−12 base pair are recognized by the σ subunit (4,18). Consistently, we found that affinity of fork junction probe 8 bearing an unpaired T at −12 was less affected by the T-12A substitution than affinities of probes 5, 6(−11T) and 9 (Figure 5). In agreement with this result, a derivative of fork junction probe 5 lacking the template strand nucleotide at position −12 (probe 31) bound RNAP ∼10-fold weaker than probe 5 (Supplementary Figure S3).Figure 5.

Bottom Line: 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 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.We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of RPo formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Waksman Institute of Microbiology Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA and Institutes of Molecular Genetics and Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.

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.

Show MeSH