Limits...
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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DNA templates used and sample EMSA. (A) Duplex is a truncated version of a longer promoter sequence used in earlier work (16). It lacks any DNA downstream of position +1. The −35 and extended −10 elements (i.e. TGXTATAAT) as well as the +1 transcription start site are shown in bold, and the −11 position is marked by a dot. Mismatch DNA contains an A, instead of a T, at −11 in the template strand, resulting in a single mismatch at this position when the base on the non-template strand is A or 2AP. The same terminology was also applied if the non-template strand contained a purine (Nebularine) or was abasic at −11. For the −11G Duplex the template strand has a −11C, and for the Mismatch −11G, a −11G. (B) The structure of Adenine and other −11 substituents used in this work. dSpacer (Integrated DNA Technologies) models an abasic site. (C) Example of an EMSA gel for reaction mixes containing radiolabeled Duplex or −11 2AP Duplex and RNAP containing WT or substituted σ70. Subsequent to incubation of RNAP and radiolabeled DNA at room temperature, the mixtures were subjected to a heparin challenge (200 μg/ml for 10 min) prior to loading onto a non-denaturing polyacrylamide gel.
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Figure 1: DNA templates used and sample EMSA. (A) Duplex is a truncated version of a longer promoter sequence used in earlier work (16). It lacks any DNA downstream of position +1. The −35 and extended −10 elements (i.e. TGXTATAAT) as well as the +1 transcription start site are shown in bold, and the −11 position is marked by a dot. Mismatch DNA contains an A, instead of a T, at −11 in the template strand, resulting in a single mismatch at this position when the base on the non-template strand is A or 2AP. The same terminology was also applied if the non-template strand contained a purine (Nebularine) or was abasic at −11. For the −11G Duplex the template strand has a −11C, and for the Mismatch −11G, a −11G. (B) The structure of Adenine and other −11 substituents used in this work. dSpacer (Integrated DNA Technologies) models an abasic site. (C) Example of an EMSA gel for reaction mixes containing radiolabeled Duplex or −11 2AP Duplex and RNAP containing WT or substituted σ70. Subsequent to incubation of RNAP and radiolabeled DNA at room temperature, the mixtures were subjected to a heparin challenge (200 μg/ml for 10 min) prior to loading onto a non-denaturing polyacrylamide gel.

Mentions: Our goal was to arrive at a better understanding of the roles of amino acid residues Y430 and W433 of σ70, and of the −11A in promoter DNA melting. Our approach has been to combine σ70 containing substitutions of A, L, F, W or H for Y430 and of A, L, F, Y or H for W433 with promoter DNAs bearing substitutions for the −11A. The DNA substrates used in this work are shown in Figure 1A. We have carried out most experiments using a promoter (‘Duplex’) that is truncated in the downstream direction at position +1, the start site of transcription. In the non-template strand these DNAs had the −11A or substitutions of 2AP, G, purine, or an abasic nucleotide (Figure 1B). Another template (‘Mismatch’) had an A at −11 in the template strand, leading to a mismatch in the templates that contained A, 2AP, or purine at −11 in the non-template strand. Duplex templates containing a G at −11 of the non-template strand had a −11C in the template strand, and a G at this position for the Mismatch DNA. The promoter used has consensus −35 and −10 regions [the latter includes the upstream TG element, and is thus an extended −10 (31,32)], to ensure tight RNAP binding in the closed complex, so that effects of substitutions would mostly be on DNA melting rather than closed complex formation. Our previous work (33) showed that substitutions at the −10 and −35 that decreased the similarity of these regions to their consensus sequences, greatly reduced binding affinity for RNAP in the closed complex. The rationale for the use of truncated templates is that the effects of substitutions in both promoter DNA and σ70 are more pronounced, as compared to a longer DNA template (to position + 20) of the same sequence (data not shown).Figure 1.


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)

DNA templates used and sample EMSA. (A) Duplex is a truncated version of a longer promoter sequence used in earlier work (16). It lacks any DNA downstream of position +1. The −35 and extended −10 elements (i.e. TGXTATAAT) as well as the +1 transcription start site are shown in bold, and the −11 position is marked by a dot. Mismatch DNA contains an A, instead of a T, at −11 in the template strand, resulting in a single mismatch at this position when the base on the non-template strand is A or 2AP. The same terminology was also applied if the non-template strand contained a purine (Nebularine) or was abasic at −11. For the −11G Duplex the template strand has a −11C, and for the Mismatch −11G, a −11G. (B) The structure of Adenine and other −11 substituents used in this work. dSpacer (Integrated DNA Technologies) models an abasic site. (C) Example of an EMSA gel for reaction mixes containing radiolabeled Duplex or −11 2AP Duplex and RNAP containing WT or substituted σ70. Subsequent to incubation of RNAP and radiolabeled DNA at room temperature, the mixtures were subjected to a heparin challenge (200 μg/ml for 10 min) prior to loading onto a non-denaturing polyacrylamide gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: DNA templates used and sample EMSA. (A) Duplex is a truncated version of a longer promoter sequence used in earlier work (16). It lacks any DNA downstream of position +1. The −35 and extended −10 elements (i.e. TGXTATAAT) as well as the +1 transcription start site are shown in bold, and the −11 position is marked by a dot. Mismatch DNA contains an A, instead of a T, at −11 in the template strand, resulting in a single mismatch at this position when the base on the non-template strand is A or 2AP. The same terminology was also applied if the non-template strand contained a purine (Nebularine) or was abasic at −11. For the −11G Duplex the template strand has a −11C, and for the Mismatch −11G, a −11G. (B) The structure of Adenine and other −11 substituents used in this work. dSpacer (Integrated DNA Technologies) models an abasic site. (C) Example of an EMSA gel for reaction mixes containing radiolabeled Duplex or −11 2AP Duplex and RNAP containing WT or substituted σ70. Subsequent to incubation of RNAP and radiolabeled DNA at room temperature, the mixtures were subjected to a heparin challenge (200 μg/ml for 10 min) prior to loading onto a non-denaturing polyacrylamide gel.
Mentions: Our goal was to arrive at a better understanding of the roles of amino acid residues Y430 and W433 of σ70, and of the −11A in promoter DNA melting. Our approach has been to combine σ70 containing substitutions of A, L, F, W or H for Y430 and of A, L, F, Y or H for W433 with promoter DNAs bearing substitutions for the −11A. The DNA substrates used in this work are shown in Figure 1A. We have carried out most experiments using a promoter (‘Duplex’) that is truncated in the downstream direction at position +1, the start site of transcription. In the non-template strand these DNAs had the −11A or substitutions of 2AP, G, purine, or an abasic nucleotide (Figure 1B). Another template (‘Mismatch’) had an A at −11 in the template strand, leading to a mismatch in the templates that contained A, 2AP, or purine at −11 in the non-template strand. Duplex templates containing a G at −11 of the non-template strand had a −11C in the template strand, and a G at this position for the Mismatch DNA. The promoter used has consensus −35 and −10 regions [the latter includes the upstream TG element, and is thus an extended −10 (31,32)], to ensure tight RNAP binding in the closed complex, so that effects of substitutions would mostly be on DNA melting rather than closed complex formation. Our previous work (33) showed that substitutions at the −10 and −35 that decreased the similarity of these regions to their consensus sequences, greatly reduced binding affinity for RNAP in the closed complex. The rationale for the use of truncated templates is that the effects of substitutions in both promoter DNA and σ70 are more pronounced, as compared to a longer DNA template (to position + 20) of the same sequence (data not shown).Figure 1.

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