Structure determination. (a) Stereo view of a portion of the 2|F_o| − |F_c| electron density map (3.2 Å, 1σ, shown in blue) calculated from the T. aquaticus core RNAP structure, showing a region corresponding to the ω subunit (including the N-terminal part of CR1 α-helix; at center, oriented horizontally) and nearby parts of β and β′. Atoms of ω are colored by atom type (C, yellow; O, red; N, blue; S, green). Atoms of β′ and β are colored pink and light blue, respectively. The SeMet difference Fourier peak (3σ) that corresponds to Met¹² of ω is shown in magenta. Selected residues of ω are labeled. The figure was generated by using the program o (40). (b) Structure of the ω subunit in T. aquaticus RNAP core enzyme. A ribbon representation of T. aquaticus ω residues (residues 2–96) is shown. Residues of ω not included in the sequence alignment in Fig. 1 are illustrated in white; conserved regions CR1–CR3 are in yellow; nonconserved regions are in cyan. S1 is part of intersubunit β-sheet (two-strand antiparallel β-sheet with residues 1483–1487 of the C-terminal tail of β′).

Bacterial ω and eukaryotic RPB6 are functional homologs. (a) Effects of overproduction of ω on the temperature-sensitive phenotypes of rpoC^tsX. The figure shows 15-h growth on LB/ampicillin plates of rpoC^tsX cells, rpoC^ts4 cells, and rpoC^397C cells, transformed with plasmids overproducing ω (pω), or a control plasmid (pUC). (Left) Permissive temperature (30°C). (Right) Nonpermissive temperature (42°C). (b) Effects of overproduction of RPB6 on the temperature-sensitive phenotypes of RPB1-1. The figure shows 48-h growth on YEPD plates of RPB1-1 cells transformed with a plasmid overproducing RPB6 (2μRPB6), or a control plasmid (2μ). (Left) Permissive temperature (30°C). (Right) Nonpermissive temperature (36°C).

Bacterial ω, archaeal RpoK, and eukaryotic RPB6 are sequence homologs. Aligned sequences of bacterial RNAP ω (Top), archaeal RNAP RpoK (Middle), and poxviral and eukaryotic RNAP RPB6 (Bottom). Residues identical in at least half of the aligned sequences and represented in all three sets of aligned sequences are in red; residues identical or similar in at least half of the aligned sequences and represented in all three sets of aligned sequences are in blue. CR1–CR3 (yellow bars) delineate conserved regions (defined as containing residues identical or similar in at least half of the aligned sequences and represented in all three sets of aligned sequences, and containing no insertions or deletions greater than one residue). Helices 2 and 3 and strand 1 in the crystallographic structure of Thermus aquaticus ω (Fig. 3) are indicated by black bars. Species names and database locus identifiers for the sequences are, in order: Escherichia coli (RPOZ_ECOLI), Haemophilus influenzae (RPOZ_HAEIN), Vibrio cholerae (AAF95849), Xylella fastidiosa (AE003980_3), Neisseria meningitidis (AAF42009), Helicobacter pylori J99 (D71898), Helicobacter pylori 26695 (H64616), Campylobacter jejuni (CAB73527), Rickettsia prowazekii (Y578_RICPR), Bradyrhizobium japonicum (AAF04326), Bacillus subtilis (C69878), Mycobacterium tuberculosis (YD90_MYCTU), Streptomyces coelicolor (CAB93358), Synechocystis sp. (Y61L_SYNY3), Treponema pallidum (A71289), Aquifex aeolicus (F70317), Deinococcus radiodurans (A75266), Thermotoga maritima (B72223), Thermus aquaticus (AJ295839; this work), Archaeoglobus fulgidus (RPOK_ARCFU), Haloarcula marismortui (RPOK_HALMA), Aeropyrum pernix (RPOK_AERPE), Methanobacterium thermoautotrophicum (RPOK_METTH), Methanococcus jannaschii (RPOK_METJA), Pyrococcus abyssi (B75172), African swine fever virus ASF M2 (RPB6_ASFM2), ASF B7 (RPB6_ASFB7), ASF 1 (S35644), Plasmodium falciparum (T18424), Saccharomyces cerevisiae RPB6_YEAST), Schizosaccharomyces pombe (RPB6_SCHPO), Nicotiana tabacum (AF153277_1), Arabidopsis thaliana AC006955_29), Caenorhabditis elegans (RPB6_CAEEL), Drosophila melanogaster (RPB6_DROME), Gallus gallus (CAB62065), Rattus norvegicus (RPB6_RAT), and Homo sapiens (RPB6_HUMAN). Chloroplast RNAP is closely related to bacterial RNAP (39); putative ω homologs are present in chloroplasts from red algae, cryptophyte algae, and low-branching green flagellates [Porphyra purpurea (YC61_PORPU), Cyanidium caldarium (YC61_CYACA), Guillardia theta (YC61_GUITH), and Mesostigma viride (AF166114–93); unpublished data]. Sequences of chloroplast putative ω homologs are highly similar to the sequence of Synechocystis sp. ω and can be aligned analogously.

Bacterial ω and eukaryotic RPB6 are structural homologs. (a) Structure of ω and the ω–β′ interface in T. aquaticus RNAP core enzyme. Residues of ω included in the sequence alignment in Fig. 1 are illustrated in a ribbon representation (residues 9–81); conserved regions CR1–CR3 are in yellow; nonconserved regions are in cyan. Residues of β′ conserved regions D and G are in pink; residues of the β′ C-terminal tail are in red, with the residue corresponding to the residue substituted in the E. coli rpoC^tsx mutant (panel f and Fig. 4a) indicated in green. (b) Location of ω within T. aquaticus RNAP core enzyme. The structure of T. aquaticus RNAP core enzyme is illustrated in a Cα representation. Conserved regions CR1–CR3 of ω are in yellow; nonconserved regions of ω are in blue; β′ is in pink, with the β′ C-terminal tail in red; β is in cyan, α^Ι and α^ΙΙ are in green; the active-center Mg²⁺ is in magenta. (c) Structure of RPB6 and the RPB6–RPB1 interface in yeast RNAP II (atomic coordinates from ref. 2, PDB accession code 1EN0, with reassignment of 79 C-terminal residues of RPB1 based on refined atomic coordinates) (P. Cramer, D. Bushnell, and R. Kornberg, personal communication). Residues of RPB6 included in the sequence alignment in Fig. 1 are illustrated in a ribbon representation (residues 80–138); conserved regions CR1–CR3 are in yellow; nonconserved regions are in cyan. Residues of RPB1 conserved regions D and G are in pink; residues of the RPB1 C-terminal tail are in red, with the residue substituted in RPB1-1 (panel f and Fig. 4b) indicated in green. (Residues of the C-terminal tail following residue Ile¹⁴⁴⁵ are not defined in the available structure.) Residue numbers in structural elements of RPB6 and RPB1 were inferred by reference to residue numbers in structurally equivalent elements of ω and β′ (a) and to sequence alignments (Fig. 1; also panel f herein), and are expected to be correct within ≈1 residue. (d) Location of RPB6 within yeast RNAP II (atomic coordinates as in c). The structure of yeast RNAP II is illustrated in a Cα representation. Conserved regions CR1–CR3 of RPB6 are in yellow; nonconserved regions of RPB6 are in blue; RPB1 is in pink, with the RPB1 C-terminal tail in red; RPB2 is in cyan, RPB3 and RPB11 are in green; subunits of RNAP II without counterparts in bacterial RNAP (RPB5, RPB8, RPB9, RPB10, and RPB12) are in gray; the active-center Mg²⁺ is in magenta. (e) Structural alignment of ω (cyan; residues 9–81) and RPB6 (red; residues 80–138). (f) Sequences of segments of the RNAP largest subunit that interact with ω (a) and RBP6 (c). Conserved regions of the RNAP largest subunit are indicated by lettered boxes (8). The amino acid substitutions in E. coli rpoC^tsX and yeast RPB1-1 are indicated above the aligned sequences. Sequences shown are, in order: T. aquaticus β′ (CAB65466), E. coli β′ (RPOC_ECOLI), S. cerevisiae RPA1 (RPA1_YEAST), S. cerevisiae RPB1 (RPB1_YEAST), and S. cerevisiae RPC1 (RPC1_YEAST). The dots indicate amino acid identities.