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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 substitutions at W433 of σ70 on stable complex formation. Determination of stable complex formation of Duplex and Mismatch DNA bearing different substitutions at −11 with RNAP containing WT or mutant σ70 with substitutions at position 433. The assay conditions are as indicated in the legend to Figure 2, and in the Materials and Methods Section. Each group of bars represents the collection of σ70 variants tested. (A) Duplex templates. (B) Mismatch templates.
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Figure 3: Effects of substitutions at W433 of σ70 on stable complex formation. Determination of stable complex formation of Duplex and Mismatch DNA bearing different substitutions at −11 with RNAP containing WT or mutant σ70 with substitutions at position 433. The assay conditions are as indicated in the legend to Figure 2, and in the Materials and Methods Section. Each group of bars represents the collection of σ70 variants tested. (A) Duplex templates. (B) Mismatch templates.

Mentions: Stable complex formation at room temperature for some combinations of promoter and σ70 substitutions are shown in Figure 1C. In this experiment, prior to loading binding reactions consisting of RNAP and labeled DNA on the gel, they were challenged for 10 min by added heparin. Heparin binds tightly to free RNAP and is a competitor with DNA for the binding to the enzyme. Due to the fact that the heparin is added in great excess, it will sequester any free RNAP that forms because of dissociation of RNAP promoter-complexes. Thus only stable complexes survive the heparin challenge. Stable complex formation between RNAP and Duplex DNA is seen to be greatly affected by the identity of the base at −11 of the non-template strand. The gel image in Figure 1C shows that stable complex formation is decreased for WT RNAP-DNA complexes containing the 2AP substitution at −11 compared to the Duplex DNA (A at −11). Figures 2 and 3 summarize the results collected for all experiments carried out with various combinations of DNA templates and RNAP bearing substitutions in σ70. The groups of bars represent different DNA templates and the bars within each group, the various amino acid substitutions. We tested purine for its lack of substituents, 2AP as an A-analog but with the amino group at the 2—instead of at the 6 position, G, which in addition to the 2-amino group also has a 6 carbonyl group, and dSpacer which has di-deoxyribose, without an attached base (Figure 1B). 2AP has found much use for its fluorescent properties (34–37), but has been a very useful probe for RNAP promoter interactions as well (19–21).Figure 2.


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 substitutions at W433 of σ70 on stable complex formation. Determination of stable complex formation of Duplex and Mismatch DNA bearing different substitutions at −11 with RNAP containing WT or mutant σ70 with substitutions at position 433. The assay conditions are as indicated in the legend to Figure 2, and in the Materials and Methods Section. Each group of bars represents the collection of σ70 variants tested. (A) Duplex templates. (B) Mismatch templates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effects of substitutions at W433 of σ70 on stable complex formation. Determination of stable complex formation of Duplex and Mismatch DNA bearing different substitutions at −11 with RNAP containing WT or mutant σ70 with substitutions at position 433. The assay conditions are as indicated in the legend to Figure 2, and in the Materials and Methods Section. Each group of bars represents the collection of σ70 variants tested. (A) Duplex templates. (B) Mismatch templates.
Mentions: Stable complex formation at room temperature for some combinations of promoter and σ70 substitutions are shown in Figure 1C. In this experiment, prior to loading binding reactions consisting of RNAP and labeled DNA on the gel, they were challenged for 10 min by added heparin. Heparin binds tightly to free RNAP and is a competitor with DNA for the binding to the enzyme. Due to the fact that the heparin is added in great excess, it will sequester any free RNAP that forms because of dissociation of RNAP promoter-complexes. Thus only stable complexes survive the heparin challenge. Stable complex formation between RNAP and Duplex DNA is seen to be greatly affected by the identity of the base at −11 of the non-template strand. The gel image in Figure 1C shows that stable complex formation is decreased for WT RNAP-DNA complexes containing the 2AP substitution at −11 compared to the Duplex DNA (A at −11). Figures 2 and 3 summarize the results collected for all experiments carried out with various combinations of DNA templates and RNAP bearing substitutions in σ70. The groups of bars represent different DNA templates and the bars within each group, the various amino acid substitutions. We tested purine for its lack of substituents, 2AP as an A-analog but with the amino group at the 2—instead of at the 6 position, G, which in addition to the 2-amino group also has a 6 carbonyl group, and dSpacer which has di-deoxyribose, without an attached base (Figure 1B). 2AP has found much use for its fluorescent properties (34–37), but has been a very useful probe for RNAP promoter interactions as well (19–21).Figure 2.

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