The Structural Basis for Promoter −35 Element Recognition by the Group IV σ Factors

Abstract

The control of bacterial transcription initiation depends on a primary σ factor for housekeeping functions, as well as alternative σ factors that control regulons in response to environmental stresses. The largest and most diverse subgroup of alternative σ factors, the group IV extracytoplasmic function σ factors, directs the transcription of genes that regulate a wide variety of responses, including envelope stress and pathogenesis. We determined the 2.3-Å resolution crystal structure of the −35 element recognition domain of a group IV σ factor, Escherichia coli σE 4, bound to its consensus −35 element, GGAACTT. Despite similar function and secondary structure, the primary and group IV σ factors recognize their −35 elements using distinct mechanisms. Conserved sequence elements of the σE −35 element induce a DNA geometry characteristic of AA/TT-tract DNA, including a rigid, straight double-helical axis and a narrow minor groove. For this reason, the highly conserved AA in the middle of the GGAACTT motif is essential for −35 element recognition by σE 4, despite the absence of direct protein–DNA interactions with these DNA bases. These principles of σE 4/−35 element recognition can be applied to a wide range of other group IV σ factors.

Binding and release of the 6S transcriptional control RNA

“…These upstream islands of sequence conservation are named the ''-35'' and ''-10'' regions after Barrick et al (2005), who noted by simple geometry that these regions loosely correspond to the location of the -35 and -10 DNA promoter elements in a DNA initiation complex. Consistent with this hypothesis, a series of protein truncation and mutation experiments have since demonstrated the importance of the s 70 4 subdomain, which has previously been shown to be responsible for -35 DNA promoter recognition in 6S RNA binding (Mooney et al 2005;Lane and Darst 2006;Cavanagh et al 2008;Klocko and Wassarman 2009). …”

The essential activities of the bacterial sigma factor

“…Factor specifically recognizes and binds the -35 hexanucleotide sequence through multiple helix-turn-helix and major groove specific and nonspecific interactions (Gardella et al 1989;Siegele et al 1989;Kenney and Moran 1991;Campbell et al 2002;Lane and Darst 2006). It was a reasonable expectation that a similarly welldefined interaction would occur between and the conserved double-stranded -10 element, although biochemical analyses have strongly implied that single-stranded non-template DNA might be a greater determinant of and -10 binding (Qiu and Helmann 1999;Fenton and Gralla 2003;Mekler and Severinov 2013).…”

Appropriate Regulation of the σ E -Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

“…The mutations decreased the specific activity of E 9-to 72-fold, with only small effects on the amount of E . These residues have been shown to form direct contacts with the Ϫ35 region of the promoter DNA, with the exception of L127, which is buried in the hydrophobic core of E domain 4 (15,29). The rpoE gene is transcribed from both 70 -and E -dependent promoters, and the slightly reduced amounts of E in these mutants could be due to reduced transcription from the E -dependent promoter or reduced stability of the protein.…”

“…3). E126 is exposed on the surface and potentially at the interface between core RNA polymerase and E , R149 and R171 make DNA contacts in the Ϫ35 promoter region, and A177 forms part of the hydrophobic core (15,29).…”

“…The L10P and A60P mutations introduce prolines in the first position of a turn and in the middle of an ␣ helix, respectively (30,31). An isoleucine substitution at position 155 likely exposes a hydrophobic residue on the surface of the protein, and V170 is buried in the hydrophobic core (15,29).…”