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

Abstract

Bacterial RNA polymerase and a “sigma” transcription factor form an initiation-competent “open” complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved -11 A-T base pair. Amino acids in conserved region 2.3 of the main E. coli sigma factor, σ70, are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-AP at −11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-AP are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of σ70 was shown to be involved in quenching the fluorescence of a 2-AP substituted at −11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase.

Antibiotics trapping transcription initiation intermediates

“…The formation of the transcription bubble starts from the nucleation of melting at the -10 element, followed by the downstream propagation toward position +1. [4][5][6] The slow isomerization from RP c to RP o involves several intermediate complexes (RP i or I 2 ) and includes large scale conformational changes in RNAP. [7][8][9][10] Kinetic studies performed primarily using the two promoters lacUV5 and lP R indicate that there is a general three-step scheme for open complex formation (Fig.…”

“…10,24 The second model, referred to as "open-bend-load," is based on real-time kinetic measurements on the T7A1 promoter 5 and on the -10/-35 synthetic consensus promoter. 6 In this model, melting precedes (or is even required for) the entry of the downstream DNA segment into the RNAP jaws. Computer-based Brownian dynamics simulations of the RP c →RP o transition have also suggested that DNA melts before entering the cleft.…”

Reduced capacity of alternative σs to melt promoters ensures stringent promoter recognition

“…We consider the implications of this finding in the Discussion. Cold (Fenton et al 2000;Tomsic et al 2001;Schroeder et al 2009). The crystal structure of Aquifex aeolicus s 28 complexed to its anti-s indicates that these Region 2.3 residues in Group 3 ss are roughly in the same position as in the housekeeping ss with respect to the most C-terminal helix of s 2 (Sorenson et al 2004), suggesting that they might play similar roles in both s families.…”

“…These activities are tightly coupled: The Region 2.3 residues facilitating melting and general duplex recognition are located in the same a-helix as the Region 2.4 residues that recognize specific bases in the À10 region and then stabilize the melted state by interaction with the nontemplate strand. In Es 70 promoters, strand separation is nucleated within the À10 recognition motif, most likely by flipping out the À11A base (Lim et al 2001;Schroeder et al 2009). W433 may ''push'' the À11A out of the helix (Tomsic et al 2001).…”

“…1A; Kuznedelov et al 2002;Murakami et al 2002). s 4 recognizes the À35 motif (Gardella et al 1989;Siegele et al 1989), s 3 recognizes the extended À10 (E-10) motif (Barne et al 1997;Koo et al 2009a,b), and s 2 facilitates strand opening by three sequential activities: (1) recognition of the À10 and discriminator regions (Siegele et al 1989;Daniels et al 1990;Waldburger et al 1990;Tatti et al 1991;Feklistov et al 2006;Haugen et al 2006;Koo et al 2009a,b), (2) participation in melting (Juang and Helmann 1994;Fenton et al 2000;Tomsic et al 2001;Lee and Gralla 2003), and (3) interaction with the À10 region nontemplate strand DNA to stabilize the melted state (Helmann and deHaseth 1999;Schroeder et al 2009). s 1 (Region 1.1) is unique to housekeeping ss and has regulatory roles (Dombroski et al 1992(Dombroski et al , 1993Hook-Barnard and Hinton 2009).…”