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The -11A of promoter DNA and two conserved amino acids in the melting region of sigma70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact.

Schroeder LA, Choi AJ, DeHaseth PL - Nucleic Acids Res. (2007)

Bottom Line: Substitutions for -11A and for Y430 and W433 in sigma70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex.As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the -11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the -11A.The effects of the substitutions at -11A of the promoter and Y430 and W433 of sigma70 are cumulative.

View Article: PubMed Central - PubMed

Affiliation: The Center for RNA Molecular Biology and The Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA. las30@case.edu

ABSTRACT
Formation of the stable, strand separated, 'open' complex between RNA polymerase and a promoter involves DNA melting of approximately 14 base pairs. The likely nucleation site is the highly conserved -11A base in the non-template strand of the -10 promoter region. Amino acid residues Y430 and W433 on the sigma70 subunit of the RNA polymerase participate in the strand separation. The roles of -11A and of the Y430 and W433 were addressed by employing synthetic consensus promoters containing base analog and other substitutions at -11 in the non-template strand, and sigma70 variants bearing amino acid substitutions at positions 430 and 433. Substitutions for -11A and for Y430 and W433 in sigma70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex. As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the -11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the -11A. The effects of the substitutions at -11A of the promoter and Y430 and W433 of sigma70 are cumulative.

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Effects of DNA and σ70 substitutions on equilibrium and kinetics of stable complex formation. (A) Kd. The asterisks above two of the bars are to indicate that these are lower estimates for the values of Kd. (B) kon (calculated from values of kobs and koff). (C) Half lives, t1/2, of the complexes, as calculated from the koff values: koff = 0.010, 0.031, 0.015 and 0.022 min−1 respectively for complexes of Duplex with WT, W433L, Y430A and Y430F RNAP, and 0.134, 0.469, 0.049 and 0.326 min−1 respectively for complexes of −11 2AP Duplex with WT, W433L, Y430A and Y430F RNAP. See the legend to Figure 5 and the Materials and Methods for experimental detail and calculation of kon and t1/2.
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Figure 6: Effects of DNA and σ70 substitutions on equilibrium and kinetics of stable complex formation. (A) Kd. The asterisks above two of the bars are to indicate that these are lower estimates for the values of Kd. (B) kon (calculated from values of kobs and koff). (C) Half lives, t1/2, of the complexes, as calculated from the koff values: koff = 0.010, 0.031, 0.015 and 0.022 min−1 respectively for complexes of Duplex with WT, W433L, Y430A and Y430F RNAP, and 0.134, 0.469, 0.049 and 0.326 min−1 respectively for complexes of −11 2AP Duplex with WT, W433L, Y430A and Y430F RNAP. See the legend to Figure 5 and the Materials and Methods for experimental detail and calculation of kon and t1/2.

Mentions: In order to try and pinpoint the step(s) in Scheme 1 at which the substitutions in promoter DNA or σ70 exert their effects, we performed binding experiments in the absence of a heparin challenge to determine whether complex formation in general (i.e. both closed and open complexes) was affected. The experiments shown in Figure 4 as well as those shown in Figures 5, 6 and 7 were carried out with a subset of promoter DNAs and σ70. The results in Figure 4A (gel image) and 4B (quantification by Phosphor Imaging) demonstrate that for both DNAs (Duplex and −11 2AP Duplex), total complex formation is similar for RNAP with WT and the three mutant σ70. If the RNAP and DNA are incubated for 30 s instead of 10 min, similar amounts of total complex formation are detected (data not shown). Thus defects in stable complex formation (Figures 1C, 2 and 3) cannot be explained by invoking effects of the promoter—or σ70 substitutions on DNA binding. Total complex formation was also not affected for RNAP binding to −11 Abasic Duplex DNA (data not shown). For the −11 G Duplex, small (less than 2-fold) differences among the various RNAP in total complex formation became apparent (data not shown), although any correlation between binding and stable complex formation is difficult to assess in view of the very low extents of stable complex formation seen with this template.Figure 4.


The -11A of promoter DNA and two conserved amino acids in the melting region of sigma70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact.

Schroeder LA, Choi AJ, DeHaseth PL - Nucleic Acids Res. (2007)

Effects of DNA and σ70 substitutions on equilibrium and kinetics of stable complex formation. (A) Kd. The asterisks above two of the bars are to indicate that these are lower estimates for the values of Kd. (B) kon (calculated from values of kobs and koff). (C) Half lives, t1/2, of the complexes, as calculated from the koff values: koff = 0.010, 0.031, 0.015 and 0.022 min−1 respectively for complexes of Duplex with WT, W433L, Y430A and Y430F RNAP, and 0.134, 0.469, 0.049 and 0.326 min−1 respectively for complexes of −11 2AP Duplex with WT, W433L, Y430A and Y430F RNAP. See the legend to Figure 5 and the Materials and Methods for experimental detail and calculation of kon and t1/2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919498&req=5

Figure 6: Effects of DNA and σ70 substitutions on equilibrium and kinetics of stable complex formation. (A) Kd. The asterisks above two of the bars are to indicate that these are lower estimates for the values of Kd. (B) kon (calculated from values of kobs and koff). (C) Half lives, t1/2, of the complexes, as calculated from the koff values: koff = 0.010, 0.031, 0.015 and 0.022 min−1 respectively for complexes of Duplex with WT, W433L, Y430A and Y430F RNAP, and 0.134, 0.469, 0.049 and 0.326 min−1 respectively for complexes of −11 2AP Duplex with WT, W433L, Y430A and Y430F RNAP. See the legend to Figure 5 and the Materials and Methods for experimental detail and calculation of kon and t1/2.
Mentions: In order to try and pinpoint the step(s) in Scheme 1 at which the substitutions in promoter DNA or σ70 exert their effects, we performed binding experiments in the absence of a heparin challenge to determine whether complex formation in general (i.e. both closed and open complexes) was affected. The experiments shown in Figure 4 as well as those shown in Figures 5, 6 and 7 were carried out with a subset of promoter DNAs and σ70. The results in Figure 4A (gel image) and 4B (quantification by Phosphor Imaging) demonstrate that for both DNAs (Duplex and −11 2AP Duplex), total complex formation is similar for RNAP with WT and the three mutant σ70. If the RNAP and DNA are incubated for 30 s instead of 10 min, similar amounts of total complex formation are detected (data not shown). Thus defects in stable complex formation (Figures 1C, 2 and 3) cannot be explained by invoking effects of the promoter—or σ70 substitutions on DNA binding. Total complex formation was also not affected for RNAP binding to −11 Abasic Duplex DNA (data not shown). For the −11 G Duplex, small (less than 2-fold) differences among the various RNAP in total complex formation became apparent (data not shown), although any correlation between binding and stable complex formation is difficult to assess in view of the very low extents of stable complex formation seen with this template.Figure 4.

Bottom Line: Substitutions for -11A and for Y430 and W433 in sigma70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex.As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the -11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the -11A.The effects of the substitutions at -11A of the promoter and Y430 and W433 of sigma70 are cumulative.

View Article: PubMed Central - PubMed

Affiliation: The Center for RNA Molecular Biology and The Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA. las30@case.edu

ABSTRACT
Formation of the stable, strand separated, 'open' complex between RNA polymerase and a promoter involves DNA melting of approximately 14 base pairs. The likely nucleation site is the highly conserved -11A base in the non-template strand of the -10 promoter region. Amino acid residues Y430 and W433 on the sigma70 subunit of the RNA polymerase participate in the strand separation. The roles of -11A and of the Y430 and W433 were addressed by employing synthetic consensus promoters containing base analog and other substitutions at -11 in the non-template strand, and sigma70 variants bearing amino acid substitutions at positions 430 and 433. Substitutions for -11A and for Y430 and W433 in sigma70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex. As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the -11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the -11A. The effects of the substitutions at -11A of the promoter and Y430 and W433 of sigma70 are cumulative.

Show MeSH
Related in: MedlinePlus