The C-Terminal RpoN Domain of sigma54 Forms an unpredicted Helix-Turn-Helix Motif Similar to domains of sigma70 (2005)

THE C-TERMINAL RPON DOMAIN OF σ 54 FORMS AN UNPREDICTED HELIX-TURN-HELIX MOTIF SIMILAR TO DOMAINS OF σ 70 Michaeleen Doucleff 1,2 , Lawrence T. Malak 1,3 , Jeffrey G. Pelton 1,2 , David E. Wemmer* 1,2 From 1 Physical Biosciences Division, Lawrence Berekeley National Laboratory, 2 Department of Chemistry, and 3 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA * Correspondence should be directed to David E. Wemmer: Physical Biosciences Divisions, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; e-mail: DEWemmer@lbl.gov; fax: (510) 486-6059. The ‘σ’ subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two σ families have been identified, σ 70 and σ 54 , which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the σ 70 -type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the σ 54 -type factors. Here we present the NMR derived structure of the C-terminal domain of σ 54 from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three- helix bundle, which is surprisingly similar to domains of the σ 70 -type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of σ 54 binds to DNA. INTRODUCTION Transcription, the synthesis of RNA from double-stranded DNA, is a fundamental process in all forms of life. The primary protein complex catalyzing transcription is composed of five subunits, α 2 β'βω, called 'core RNA polymerase (RNAP)'. Core RNAP is fully competent to synthesize RNA from DNA. However, the initiation of transcription at specific DNA sequences requires additional protein(s). In bacteria, these additional proteins are called the 'σ factors' (1, 2). The roles of the σ factor are three fold: 1. Bind to the core RNAP to form the σ-RNAP holoenzyme; 2. Recognize and bind to a specific DNA sequence next to the transcription start site, called the promoter element; 3. Facilitate the initiation of transcription by opening the double-stranded DNA. Based on protein sequence homology, σ factors can be grouped into two classes, σ 70 and σ 54 , which were named by the molecular weights of the first members identified (reviewed in 3). The σ 70 -type factors are the most abundant in bacteria (reviewed in 4, 5). They include the primary σ factors, such as σ 70 from Escherichia coli and σ A from Gram-positive bacteria, which regulate transcription for most genes required for normal exponential growth. Other members of the σ 70 family (σ 28 , σ 32 , et cetera) regulate the transcription of more specialized genes that are required to respond to environmental changes. For example, σ28 from Salmonella typhimurium controls expression of genes required for flagellar assembly (6). The σ -type factor, which is also called σ Ν and encoded by the rpoN gene, has no detectable sequence homology to σ 70 -type factors. Its occurrence is widespread among bacteria, but there is usually only one σ 54 gene present in a particular organism (7). σ regulates gene transcription for proteins required for many important cellular functions, such as nitrogen metabolism, chemotaxis, and heat and phage shock responses. Recently, σ has been shown to be required for mammalian infection by Borrelia burgdorferi, the agent of Lyme disease (8). The most striking difference between σ and σ 70 factors is how they regulate transcription