Amino-terminal sequences of ςN (ς54) inhibit RNA polymerase isomerization

RNA polymerases (RNAPs) are key targets for transcriptional activators that enable processes such as cellular differentiation and adaptation to take place in an ordered manner. Regulated and specific initiation of transcription in bacteria requires a σ protein, which binds to a single common core RNAP (α2ββ′) to form a holoenzyme that directs specific initiation of RNA synthesis (Gross et al. 1992). The major σ class members are similar to the primary Escherichia coli σ70 (Lonetto et al. 1992). The σN class is unrelated (Merrick 1993), and the events leading to initiation at σN-dependent promoters are clearly distinct from those for σ70-dependent initiation. The σN-holoenzyme is required for enhancer-dependent transcription, characterized as depending on activator γ–β bond hydrolysis of a nucleoside triphosphate (Kustu et al. 1989; Weiss et al. 1991; Austin and Dixon 1992). Activators of the σN-holoenzyme belong to a chaperone-like ATPase protein family, members of which function as oligomers to remodel their substrates in a nucleotide-dependent manner in processes such as transcription, DNA replication, recombination, and restriction (Confalonieri and Duguet 1995; Swaffield et al. 1995; Wyman et al. 1997; Rippe et al. 1998). Occupancy of promoters by the σN-holoenzyme is not limiting for transcription and occurs largely independently of activator (Popham et al. 1991; Wang et al. 1997b) in contrast to other RNA polymerases that are recruited to the promoter (for review, see Ptashne and Gann 1997). The σN-holoenzyme binds promoters in a transcriptionally inactive complex (Sasse-Dwight and Gralla 1988; Kustu et al. 1989; Popham et al. 1989; Lee and Hoover 1995; Wedel and Kustu 1995) without closely contacting the transcription start site (Popham et al. 1991; Cannon et al. 1995a). Because σN interacts with sequences that become melted during activation, σN may participate in DNA melting, acting either positively or negatively (Buck and Cannon 1992; Cannon et al. 1995a; Casaz and Buck 1997; Oguiza and Buck 1997). The σN domain responsible for promoter contact is at the carboxyl terminus, and sequences that promote activation are in the amino-terminal Region I (Merrick 1993; Cannon et al. 1994, 1995b; Wong et al. 1994; Guo and Gralla 1997; Wang et al. 1997a). Region I has a negative function demonstrated by mutants that allow increased activator-independent transcription in vitro (Wang et al. 1997a). Only the holoenzyme form of RNAP makes specific open promoter complexes on double-stranded DNA (Burgess et al. 1969; Helmann and Chamberlin 1988). The σ70-holoenzyme binds promoters in a transcriptionally active form, and σ70 has a role in DNA melting through contact made with the nontemplate melted DNA strand (for review, see Chan et al. 1996). Current models of activation of the σN-holoenzyme suggest that both kinetic and thermodynamic barriers to open complex formation are overcome by activators (Wedel and Kustu 1995) and that the Region I of σN inhibits the melting function of the holoenzyme (Wang et al. 1997a). Holoenzymes that lack Region I transcribe by use of an unstable intermediate associated with transient DNA melting (Wang et al. 1997a). Protein footprints have shown that the relationship of Region I to core RNAP changes when the holoenzyme is activated, suggesting that changes in Region I may facilitate steps of the activation event (Casaz and Buck 1997). Melting of DNA is an important regulated activity in processes such as transcription and replication. In this paper, we provide evidence that formation of σN-holoenzyme pre-initiation complexes that are stably bound to melted DNA sequences requires an activator-driven conformational change in the holoenzyme. The σN-holoenzyme isomerization, normally inhibited by Region I, leads to an unmasking of a single-strand DNA-binding activity in the holoenzyme. These results explain how holoenzymes lacking Region I can transcribe without activator under solution conditions favoring transient DNA melting (Wang et al. 1997a) and why efficient and faithful transcription from pre-melted DNA templates still requires activator (Wedel and Kustu 1995). We propose that activators of the σN-holoenzyme act to facilitate a changing interaction between Region I and core to permit polymerase isomerization and allow the subsequent engagement of the holoenzyme with melted DNA so as to form a stable open promoter complex.