Limits...
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.

Show MeSH
Effect of the T−7A substitution on RNAP binding to fork junction probes. The sequences of double-stranded parts of the probes are shown in Figure 1A. Non-consensus −10 element bases are shown in italic.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3753650&req=5

gkt541-F2: Effect of the T−7A substitution on RNAP binding to fork junction probes. The sequences of double-stranded parts of the probes are shown in Figure 1A. Non-consensus −10 element bases are shown in italic.

Mentions: The fact that the introduction of −7T confers a ∼120-fold improvement in affinity of −11T probes [compare Kd values for probes 5(−11T) and 6(−11T) in Figure 1C] demonstrates that specific interaction of −11A with RNAP is not strictly required for the recognition of −7T in the context of fork junctions. We evaluated the specificity of RNAP interaction with −7T in the context of several fork junctions with non-consensus bases at positions −11 to −8 (Figure 2). Introduction of A at position −7 caused, respectively, 210- and 710-fold drops in affinity of probes 8 and 9 with consensus −10 element. A 110-fold drop was observed in the context of a probe with a non-consensus -11T [probe 6(−11T)]. A much lower, ∼8-fold, effect was observed with probe 10 bearing a G−10T−9T−8 non-consensus base stretch. Finally, the T−7A substitution caused only a 2.4-fold effect in the context of fork junction 11 with non-consensus bases at positions −11 to −8. Thus, the efficiency of −7T recognition is clearly modulated by the strength of RNAP contacts with other −10 element bases.Figure 2.


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−7A substitution on RNAP binding to fork junction probes. The sequences of double-stranded parts of the probes are shown in Figure 1A. Non-consensus −10 element bases are shown in italic.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt541-F2: Effect of the T−7A substitution on RNAP binding to fork junction probes. The sequences of double-stranded parts of the probes are shown in Figure 1A. Non-consensus −10 element bases are shown in italic.
Mentions: The fact that the introduction of −7T confers a ∼120-fold improvement in affinity of −11T probes [compare Kd values for probes 5(−11T) and 6(−11T) in Figure 1C] demonstrates that specific interaction of −11A with RNAP is not strictly required for the recognition of −7T in the context of fork junctions. We evaluated the specificity of RNAP interaction with −7T in the context of several fork junctions with non-consensus bases at positions −11 to −8 (Figure 2). Introduction of A at position −7 caused, respectively, 210- and 710-fold drops in affinity of probes 8 and 9 with consensus −10 element. A 110-fold drop was observed in the context of a probe with a non-consensus -11T [probe 6(−11T)]. A much lower, ∼8-fold, effect was observed with probe 10 bearing a G−10T−9T−8 non-consensus base stretch. Finally, the T−7A substitution caused only a 2.4-fold effect in the context of fork junction 11 with non-consensus bases at positions −11 to −8. Thus, the efficiency of −7T recognition is clearly modulated by the strength of RNAP contacts with other −10 element bases.Figure 2.

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