Threonine 429 of Escherichia coli σ70 is a Key Participant in Promoter DNA Melting by RNA Polymerase

Abstract

Initiation of transcription is an important target for regulation of gene expression. In bacteria, the formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of Escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for threonine (T) at position 429 of sigma(70): its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by T429A substitution, thus providing evidence for a role of T429 in the strand-separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding that stabilizes the highly conserved -11A-T base pairs of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the -10 region. This model suggests an active role for RNA polymerase in disrupting the -11 base pair, rather than just capturing the -11A subsequent to spontaneous unpairing.

Structural Basis for Promoter −10 Element Recognition by the Bacterial RNA Polymerase σ Subunit

“…Of all the highly conserved residues of σ region 2.3 (Figure 1C), T252 is the least tolerant to substitution (Waldburger and Susskind, 1994). Changes at this position yield the most severe promoter melting defects in vitro (Schroeder et al, 2008), and the only substitution that yields functional σ in vivo is highly conservative S (Waldburger and Susskind, 1994). Comparison of promoter binding vs. melting activities of the E. coli σ 70 T429A substitution (corresponding to Taq σ A T252A) suggests that T252 exerts its critical role at the strand separation step, after formation of RP c (Schroeder et al, 2008), consistent with the structure.…”

“…Many σ 2 residues implicated previously in promoter binding [R237, K241 (Tomsic et al, 2001); K249 (Waldburger and Susskind, 1994); R259 (Fenton et al, 2000)] or promoter-melting [F248, Y253, W256, W257 (Juang and Helmann, 1994; Schroeder et al, 2009); T252 (Schroeder et al, 2008)] are seen to play important roles in the complex. The conserved aromatic residues of σ region 2.3 were presumed to fulfill their promoter melting role through stacking interactions with the –10 element nucleotide bases (Helmann and Chamberlin, 1988), but only Y253 participates in the complex in this way by stacking on the flipped-out A -11 (Figures 2B, 3B; (Schroeder et al, 2009).…”

Evidence for a Tyrosine–Adenine Stacking Interaction and for a Short-lived Open Intermediate Subsequent to Initial Binding of Escherichia coli RNA Polymerase to Promoter DNA

“…To examine DNA opening without complications due to effects on initial binding of RNAP, promoter DNA substrates have the consensus core promoter sequences at the −35 and extended −10 regions (figure 1a 20; 21; 22). This 65 bp DNA oligomer was designed to favor tight binding, compensating for any disruptive substitutions of 2-AP and/or amino acids in region 2.3 of σ 70 , as shown below.…”

“…This 65 bp DNA oligomer was designed to favor tight binding, compensating for any disruptive substitutions of 2-AP and/or amino acids in region 2.3 of σ 70 , as shown below. The holo RNAP contained either WT σ 70 , or σ 70 with the single alanine substitutions for F427, T429, Y430, W433 and W434, region 2.3 residues that play a role in the RNAP-induced promoter strand separation 14; 15; 16; 21; 23. RNAP containing multiple substitutions in σ 70 were also investigated: YW has both the Y430A and W433A substitutions, FYW additionally has F427A, and FYWW additionally W434A.…”

Promoter spacer DNA plays an active role in integrating the functional consequences of RNA polymerase contacts with −10 and −35 promoter elements

“…This adenine is recognized specifically by RNA polymerase which targets N1 position of the adenine [26]. Thr429 has been suggested to be involved in N1 position recognition [27]. This is significant since N1 position is involved in hydrogen bonding in an A/T base pair.…”