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
RNAP binding to promoter fragments bearing −10 element-template strand bases. (A) Calculated Kd values. The sequence of −38 to −12 segment of the probes corresponds to that of probe 1. (B) Inhibitory effect of ds segment bearing non-consensus −10 element bases (shown in italic) on RNAP binding to promoter fragment. The sequence of probe 24 is shown on the top of the panel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt541-F6: RNAP binding to promoter fragments bearing −10 element-template strand bases. (A) Calculated Kd values. The sequence of −38 to −12 segment of the probes corresponds to that of probe 1. (B) Inhibitory effect of ds segment bearing non-consensus −10 element bases (shown in italic) on RNAP binding to promoter fragment. The sequence of probe 24 is shown on the top of the panel.

Mentions: Based on the aforementioned results, we created a set of ds and fork junction probes (probes 19–25, 27–29; Figure 6 and Supplementary Figure S1) bearing t-strand nucleotides downstream from the −12 position and measured affinities of these probes to RNAP. In the context of progressively extended ds probes 19–23, the introduction of the −10T/A bp resulted in inhibition of RNAP binding (Figure 6A), similarly to what was observed with fork junction probes. As expected, the A−11T substitution strongly affected affinity of ds probes. Probe 23(−11T) extended to −7 binds RNAP only 4-fold stronger than probe 1 bearing no nucleotides downstream from −12, whereas probe 22(−11T) with downstream end at −8 binds RNAP even weaker than probe 1 (Kd values are 16, 62 and 120 nM, respectively, Figures 1C and 6A). We further examined the effect of a ds segment bearing non-consensus −10 element bases in the context of probes 24 and 25 (Figure 6B) containing a sequence upstream of the −35 element which interacts effectively with the RNAP α subunit C-terminal domain (34) and a TG motif of extended −10 element. The data show that introduction of four non-consensus base pairs downstream from the −12 position in probe 24 leads to a ∼50-fold decrease in the affinity (Figure 6B). In contrast, fork junction derivatives of probe 24 containing either t-strand or nt-strand non-consensus bases bind RNAP stronger than the parent probe 24 (Supplementary Figure S4).Figure 6.


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)

RNAP binding to promoter fragments bearing −10 element-template strand bases. (A) Calculated Kd values. The sequence of −38 to −12 segment of the probes corresponds to that of probe 1. (B) Inhibitory effect of ds segment bearing non-consensus −10 element bases (shown in italic) on RNAP binding to promoter fragment. The sequence of probe 24 is shown on the top of the panel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt541-F6: RNAP binding to promoter fragments bearing −10 element-template strand bases. (A) Calculated Kd values. The sequence of −38 to −12 segment of the probes corresponds to that of probe 1. (B) Inhibitory effect of ds segment bearing non-consensus −10 element bases (shown in italic) on RNAP binding to promoter fragment. The sequence of probe 24 is shown on the top of the panel.
Mentions: Based on the aforementioned results, we created a set of ds and fork junction probes (probes 19–25, 27–29; Figure 6 and Supplementary Figure S1) bearing t-strand nucleotides downstream from the −12 position and measured affinities of these probes to RNAP. In the context of progressively extended ds probes 19–23, the introduction of the −10T/A bp resulted in inhibition of RNAP binding (Figure 6A), similarly to what was observed with fork junction probes. As expected, the A−11T substitution strongly affected affinity of ds probes. Probe 23(−11T) extended to −7 binds RNAP only 4-fold stronger than probe 1 bearing no nucleotides downstream from −12, whereas probe 22(−11T) with downstream end at −8 binds RNAP even weaker than probe 1 (Kd values are 16, 62 and 120 nM, respectively, Figures 1C and 6A). We further examined the effect of a ds segment bearing non-consensus −10 element bases in the context of probes 24 and 25 (Figure 6B) containing a sequence upstream of the −35 element which interacts effectively with the RNAP α subunit C-terminal domain (34) and a TG motif of extended −10 element. The data show that introduction of four non-consensus base pairs downstream from the −12 position in probe 24 leads to a ∼50-fold decrease in the affinity (Figure 6B). In contrast, fork junction derivatives of probe 24 containing either t-strand or nt-strand non-consensus bases bind RNAP stronger than the parent probe 24 (Supplementary Figure S4).Figure 6.

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