Structural basis of ECF-σ-factor-dependent transcription initiation

Abstract

Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σ^L, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor-although shorter and unrelated in sequence-follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent transcription initiation.

Fig. 1

Structures of group-1 and ECF σ factors. a Structural organization of group-1 (σ^A) and ECF (σ^L) σ factors. Conserved regions σR1.1, σR1.2, σR2, σR3, σR3/4 linker, and σR4 are in light orange, dark orange, yellow, green, dark green, and cyan, respectively. Dashed line, non-conserved σR2/4 linker present in ECF σ factors. b Crystal structures of group-1 (Mtb RPitc-σ^A; PDB 5UH8) and ECF (Mtb RPitc-σ^L; PDB 6DVC) transcription initiation complexes (two orthogonal views of each). σ factors are shown in tube representations with conserved regions colored as in (a). Gray ribbon, RNAP core enzyme; blue, pink, red, and magenta ribbons, -10 element of DNA nontemplate strand, rest of DNA nontemplate strand, DNA template strand, and RNA product; violet sphere, RNAP active-center Mg²⁺. Other colors are as in (a). See Supplementary Figs. 1, 2, and 7

Fig. 2

Protein–protein interactions between group-1 and ECF σ factors and RNAP core enzyme. a Protein–protein interactions by group-1 (σ^A) σ factor. b Protein–protein interactions by ECF (σ^L) σ factor. Colors are as in Fig. 1. See Supplementary Fig. 7

Fig. 3

Protein–nucleic acid interactions by group-1 and ECF σ factors: summary. a Summary of protein–nucleic acid interactions in Mtb RPitc-σ^A. Black residue numbers and lines, interactions by Mtb RNAP; green residue numbers and lines, interactions by Mtb σ^A; blue, -10 element of DNA nontemplate strand; light blue, discriminator element of DNA nontemplate strand; pink, rest of DNA nontemplate strand; red, DNA template strand; magenta, RNA product; violet circle, RNAP active-center Mg²⁺; cyan boxes, bases unstacked and inserted into protein pockets. Residues are numbered as in Mtb RNAP. b Summary of protein–nucleic acid interactions in Mtb RPitc-σ^L. Green residue numbers and lines, interactions by Mtb σ^L. Other colors are as in (a). See Supplementary Figs. 3–5 and 7

Fig. 4

Protein–nucleic acid interactions by group-1 and ECF σ factors: interactions with transcription bubble. a Left: interactions of Mtb RNAP and σ^A with transcription-bubble nontemplate strand, transcription-bubble template strand, and downstream dsDNA. Right: interactions of σ^A σR2 with σ^A-dependent promoter -10 element. For promoter positions -11 (“master nucleotide”) and -7, bases are unstacked and inserted into pockets (cyan boxes). Colors are as in Figs. 1–3. b Left: interactions of Mtb RNAP and σ^L with transcription-bubble nontemplate strand, transcription-bubble template strand, and downstream dsDNA. Right: interactions of σ^L σR2 with σ^L-dependent promoter -10 element. For two promoter positions, here designated “-11” (“master nucleotide”) and “-7”, by analogy to corresponding nucleotides in group-1-σ-factor complex (panel a), bases are unstacked and inserted into pockets (cyan boxes). For one additional nucleotide, here designated “-12”, the base also appears to be unstacked and inserted into a pocket (dashed cyan box). See Supplementary Figs. 4, 5 and 7

Fig. 5

Recognition by Mtb σ^L of σ^L-promoter -10 element: interactions with nontemplate strand positions “-12” through “-7”. For each promoter position, left subpanel shows DNA nucleotides in stick representation to highlight individual protein-nucleotide interactions, and right subpanel shows DNA nucleotide base moieties in space-filling representation to highlight protein-base steric complementarity. Yellow surfaces, solvent-accessible surfaces of Mtb σ^L σR2; gray surfaces, solvent-accessible surfaces of Mtb RNAP β subunit; dark blue surfaces, van der Waals surfaces of bases of σ^L-promoter -10 element; yellow ribbons, Mtb σ^L σR2 backbone; gray ribbons, Mtb RNAP β subunit backbone; yellow, yellow-blue, and yellow-red stick representations, σ^L carbon, nitrogen, and oxygen atoms, respectively; white, blue, red, and orange stick representations, DNA carbon, nitrogen, oxygen, and phosphorous atoms, respectively; blue dashed lines, H-bonds. Residues are numbered as in Mtb RNAP and σ^L, and, in parentheses, as in E. coli RNAP and σ⁷⁰. See Supplementary Fig. 4

Fig. 6

Recognition by Mtb σ^L of σ^L-promoter -10 element: experimental data. a Systematic-substitution experiments defining σ^L-dependent promoter -10-element consensus sequence. Relative transcriptional activities of derivatives of σ^L-dependent promoter P-sigL having all possible single base-pair substitutions at each position of promoter -10 element, “-12” through “-7”. Inferred consensus nucleotides are shown at the bottom, and data for inferred consensus nucleotides are hatched. Error bars, SE (N = 3). b Sequence logo for σ^L-promoter -10-element consensus sequence [generated using transcription data from (a) and enoLOGOS (http://biodev.hgen.pitt.edu/enologos/); input setting “energy (2)” and weight-type setting “probabilities”]. c Alanine-scanning experiments demonstrating functional importance of observed amino acid-base interactions in recognition of σ^L-promoter -10 element. Effects on transcription of alanine substitutions of σ^L amino acids that contact σ^L-dependent promoter -10 element, positions “-12” through “-7” (identities of contacting amino acids from Figs. 3 and 5). d, e Loss-of-contact experiments^– indicating that σ^L residues His54 and Asp60 determine specificity at position “-12” and “-11”, respectively. Left: transcriptional activity with wild-type σ^L for all possible single base-pair-substitutions at indicated position (strong specificity for consensus base pair). Right: transcriptional activity of σ^L derivatives having alanine substitutions (no specificity for consensus base pair). Error bars, SE (N = 3). See Supplementary Fig. 6. Source data are provided as a Source Data file