Abstract:Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the A subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75°C. However, homologous substitutions in 70 render E.… Show more
“…Remarkably, the effect of the G424R substitution on promoter opening has been observed only in combination with substitutions in subregions 2.1 and 2.2. In agreement with this, it has been reported that a single complementary R247G substitution in T. aquaticus A did not affect promoter binding by T. aquaticus RNAP holoenzyme (19). At the same time, this substitution enhanced RNAP interactions with single-stranded DNA, suggesting a role for this amino acid in promoter opening (19).…”
Highly conserved amino acid residues in region 2 of the RNA polymerase subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.Promoter opening is a temperature-dependent process that requires conformational changes in both RNA polymerase (RNAP) and DNA (4). In bacteria, promoter recognition and opening are accomplished by the RNAP holoenzyme, which consists of a core enzyme and a specificity factor, the subunit. DNA melting is initiated through specific interactions of conserved region 2 of the subunit with the Ϫ10 promoter element (consensus sequence, TATAAT) (7). Genetic and biochemical analyses have implicated conserved amino acids from different subregions of region 2 both in interactions with core RNAP and in DNA melting. Mutations of conserved residues in subregions 2.1 and 2.2 affected -core interactions and defined this part of region 2 as the main core binding site of ( Fig. 1A) (20). Mutations in a cluster of highly conserved aromatic residues in subregion 2.3 of Escherichia coli 70 and
Bacillus subtilisA were shown to lead to severe defects in promoter opening that could be partially suppressed by an increase in the temperature (Fig. 1A) (6, 9, 16). It was therefore proposed that these amino acids may directly initiate DNA melting through hydrophobic interactions with nucleotide bases of the Ϫ10 element. Particular attention has been paid to Y430 and W433 (E. coli numbering), which were proposed to interact with a conserved adenine at the second position of the Ϫ10 element (position Ϫ11A) in the nontemplate DNA strand (reference 18 and references therein). Mutations of other conserved residues in subregions 2.2, 2.3, and 2.4 (including basic amino acids in subregion 2.3) also had dramatic effects on promoter opening (Fig. 1A). These amino acids were proposed to play a role in the recognition and/or proper positioning of promoter DNA (6, 7, 21). At the same time, the detailed mechanism of promoter melting remains unknown due to the absence of information on the high-resolution structure of the open promoter complex.In RNAPs from different species there are certain functional differences in promoter recognition and opening which are believed to result, at least in part, from their different adaptations (1, 3). In particular, RNAPs from thermophilic bacteria have long been known to melt DNA and initiate transcription at higher temperatures than their mesophilic counterparts (12,13,15,17,24). In accordance with this, we recently demonstrated that RNAP from the thermophilic bacterium Thermus aquaticus opens promoters at temperatures above 37°C but, unlike RNAP from E. coli, is unable to open promoters at lower temperatures (10). Surprisingly, we found that RNAP from the mesophilic organism Deinococcus radiodurans, which is closely related phylogenetically to T. aquaticus, had similar cold sensitivity of promoter opening, implying that this prope...
“…Remarkably, the effect of the G424R substitution on promoter opening has been observed only in combination with substitutions in subregions 2.1 and 2.2. In agreement with this, it has been reported that a single complementary R247G substitution in T. aquaticus A did not affect promoter binding by T. aquaticus RNAP holoenzyme (19). At the same time, this substitution enhanced RNAP interactions with single-stranded DNA, suggesting a role for this amino acid in promoter opening (19).…”
Highly conserved amino acid residues in region 2 of the RNA polymerase subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.Promoter opening is a temperature-dependent process that requires conformational changes in both RNA polymerase (RNAP) and DNA (4). In bacteria, promoter recognition and opening are accomplished by the RNAP holoenzyme, which consists of a core enzyme and a specificity factor, the subunit. DNA melting is initiated through specific interactions of conserved region 2 of the subunit with the Ϫ10 promoter element (consensus sequence, TATAAT) (7). Genetic and biochemical analyses have implicated conserved amino acids from different subregions of region 2 both in interactions with core RNAP and in DNA melting. Mutations of conserved residues in subregions 2.1 and 2.2 affected -core interactions and defined this part of region 2 as the main core binding site of ( Fig. 1A) (20). Mutations in a cluster of highly conserved aromatic residues in subregion 2.3 of Escherichia coli 70 and
Bacillus subtilisA were shown to lead to severe defects in promoter opening that could be partially suppressed by an increase in the temperature (Fig. 1A) (6, 9, 16). It was therefore proposed that these amino acids may directly initiate DNA melting through hydrophobic interactions with nucleotide bases of the Ϫ10 element. Particular attention has been paid to Y430 and W433 (E. coli numbering), which were proposed to interact with a conserved adenine at the second position of the Ϫ10 element (position Ϫ11A) in the nontemplate DNA strand (reference 18 and references therein). Mutations of other conserved residues in subregions 2.2, 2.3, and 2.4 (including basic amino acids in subregion 2.3) also had dramatic effects on promoter opening (Fig. 1A). These amino acids were proposed to play a role in the recognition and/or proper positioning of promoter DNA (6, 7, 21). At the same time, the detailed mechanism of promoter melting remains unknown due to the absence of information on the high-resolution structure of the open promoter complex.In RNAPs from different species there are certain functional differences in promoter recognition and opening which are believed to result, at least in part, from their different adaptations (1, 3). In particular, RNAPs from thermophilic bacteria have long been known to melt DNA and initiate transcription at higher temperatures than their mesophilic counterparts (12,13,15,17,24). In accordance with this, we recently demonstrated that RNAP from the thermophilic bacterium Thermus aquaticus opens promoters at temperatures above 37°C but, unlike RNAP from E. coli, is unable to open promoters at lower temperatures (10). Surprisingly, we found that RNAP from the mesophilic organism Deinococcus radiodurans, which is closely related phylogenetically to T. aquaticus, had similar cold sensitivity of promoter opening, implying that this prope...
“…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.…”
Section: Resultsmentioning
confidence: 99%
“…Substitutions at both positions have been found to be deleterious for promoter DNA melting (10,11,13,14), but remarkably, the Y430A substitution was also found to facilitate formation of complexes of RNAP with short model templates at low temperatures (15), suggesting an inhibitory role for the Y430 residue under some circumstances. The effects of substitutions for Y430 and W433 have been found to be cumulative (11,12,16). A substantial amount of experimental evidence supports a crucial role for the − 11A base on the non-template strand in initiating the process of promoter DNA strand separation (17–23), but not in formation of the closed complex (17).…”
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 σ70 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 σ70 variants bearing amino acid substitutions at positions 430 and 433. Substitutions for −11A and for Y430 and W433 in σ70 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 σ70 are cumulative.
“…The KMnO 4 probing protocol was exactly as described (19). Separation of the fragments generated from the unmodified DNA was accomplished by running the samples on a 10% sequencing gel.…”
Formation of the strand-separated, open complex between RNA polymerase and a promoter involves several intermediates, the first being the closed complex in which the DNA is fully base-paired. This normally short lived complex has been difficult to study. We have used a mutant Escherichia coli RNA polymerase, deficient in promoter DNA melting, and variants of the P R promoter of bacteriophage to model the closed complex intermediate at physiologically relevant temperatures. Our results indicate that in the closed complex, RNA polymerase recognizes base pairs as double-stranded DNA even in the region that becomes single-stranded in the open complex. Additionally, a particular base pair in the ؊35 region engages in an important interaction with the RNA polymerase, and a DNase I-hypersensitive site, pronounced in the promoter DNA of the open complex, was not present. The effect of temperature on closed complex formation was found to be small over the temperature range from 15 to 37°C. This suggests that low temperature complexes of wild type RNA polymerase and promoter DNA may adequately model the closed complex.Bacterial RNA polymerase (RNAP) 2 that is able to engage in formation of a functionally competent complex at a promoter consists of the multisubunit core enzyme and the 70 initiation factor, which imparts on the enzyme the ability to both recognize specific DNA sequence and to melt promoter DNA (1, 2). Formation of a functional complex at a promoter is a multistep process (3) involving several intermediates. Subsequent to formation of the first "closed" RNAP-promoter complex, several rearrangements take place, involving changes in the conformations of both the RNAP and the promoter DNA (3-7), eventually resulting in formation of the transcriptionally competent open complex (RP o ) in which strand separation has occurred over about 14 bp of promoter DNA (6 -8); see Scheme 1,where RP c is the closed complex; RP c and I 1 are both unstable intermediates, I 2 is stable, and RP o is the stable final, transcriptionally competent, complex. The latter two complexes have a long half-life, rendering them resistant to heparin, a competitor with promoter DNA for RNAP binding. The rate-limiting step is the I 1 to I 2 conversion (6, 7). To fully understand the reaction pathway, it is important to know in detail the properties of the intermediate complexes.Record and co-workers (6, 7) have altered solution conditions and reaction times to favor either I 1 or I 2 . Thus they were able to determine that for the unstable I 1 complex, RNAP covers the DNA in the downstream direction up to about ϩ19 (9). In I 2 , the RNAP covers the DNA over a similarly long stretch as in I 1 . It has been proposed, although not yet experimentally verified, that in I 2 the nucleation of the strand separation process has occurred (6, 7). The closed complex is very short lived at most promoters. Attempts to arrest the reaction pathway (see Scheme 1) at RP c have involved resorting to low temperature incubation (10, 11), working with promoters fo...
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