Conserved regions in Rpo41p and T7 RNAP. (A) Sequence alignment of Rpo41p and T7 RNAP. Vertical black bars represent eight highly conserved regions between Rpo41p and T7 RNAP (T7 RNAP aa 316–337, 351–378, 802–817; Masters et al., 1987); grey bars correspond to additional regions with limited similarity. The width of the bars and the distances between them are scaled to the primary sequences of the corresponding proteins. Arrows indicate the relative positions of His-tag insertions and the names of corresponding mutants. The striped box at the N-terminus of Rpo41p indicates the relative size and position of the predicted mt targeting peptide (Wang and Shadel, 1999). (B) Alignments of targeted regions in Rpo41p. Sequences within regions of high conservation (black background) and limited similarity (grey background) are numbered as previously described. Amino acid residues are shown in one-letter code; dots represent identical residues; dashes indicate possible deletions. Pairs of residues between which His-tag insertions were introduced are indicated with asterisks. (C) Positions of His-tag insertions mapped onto the T7 RNAP-promoter structure (modified from PDB 1CEZ; Cheetham et al., 1999). The α carbon backbone of the T7 RNAP is represented as a ribbon cartoon; segments in yellow indicate regions that are highly conserved in Rpo41p (aa 142–150, 270–286, 402–483, 501–517, 537–575, 626–640, 725–740, 771–790); the region in green corresponds to the specificity loop (aa 739–770) that is responsible for promoter recognition (Temiakov et al., 2000). DNA template and non-template strands are red and blue, respectively. Aspartic acid residues 537 and 812, which coordinate Mg²⁺ ions at the active centre and are conserved among all T7-like RNAPs, are shown in magenta. Positions where His-tags were inserted in the corresponding regions of Rpo41p are indicated with red arrows and the name of the mutant. N- and C-termini are marked with white letters; note that the highly conserved C-terminus is located within or near the active site

Respiratory activity of insertion mutants of Rpo41p in vivo. The chromosomal RPO41 gene was replaced with a number of mutant genes containing insertions by using a plasmid-shuffling technique (see Materials and methods), and the ability of the resulting strains to respire was determined by plating on glycerol-containing media (YPGly) under normal (30 °C) and heat-shock conditions (37 °C) (YPD, YP w/dextrose; YPGly, YP w/glycerol; CFU, colony-forming units at appropriate dilutions). ‘315’ indicates a respiratory-defective strain (ρ⁰) used as control. Respiratory activities of EI (A), other His-tag insertion mutants (B) and N-terminal Rpo41p–TAP fusion mutant (N-TAP) (C) were determined in a similar manner. Strains expressing plasmid-encoded N-TAP-tagged and WT Rpo41p exhibited no difference in growth in a liquid medium with a non-fermentable carbon source (YP/galactose), while a strain harbouring a His-tag insertion in an alternative position (EI) did (D)

Isolation of active mt nucleoids and identification of proteins associated with TAP–mtRNAP. (A) Purification of transcriptionally active mt nucleoids. Nucleoids were purified as described (see Materials and methods) and diluted as indicated. Transcription reactions were carried out for 20 min at 30 °C in the presence of 20 or 200 mm KCl or α-amanitin (AM). The labelled RNA products were resolved by electrophoresis in a 12% acrylamide denaturing gel, visualized by autoradiography and quantified by PhosphorImager analysis. The abundance of heterogeneous high molecular weight products under various conditions is shown; error bars represent standard error for mt nucleoid transcripts (n = 3). (B) Rpo41p–TAP-associated proteins. Peptides eluted from the calmodulin–sepharose column were resolved on 4–12% bis-Tris NuPAGE Novex protein gels (Invitrogen) and visualized by silver staining (lane CE). Numbers indicate the molecular weights of protein markers in kDa (lane M). The bands from lane CE were submitted for LC–MS–MS analysis (Tufts University Core Facility). Arrows indicate silver-stained bands that match the molecular weight (MW) of major proteins identified in the gel slabs containing the bands. *Note that the actual MW of mature Pet127p may be smaller than predicted from its ORF as ∼90 N-terminal aa are likely to be absent in the mature form of Pet127p (Wiesenberger and Fox, 1997). (C) Example of LC–MS–MS spectra (Mss116p). Eleven peptides matching the Mss116p protein sequence were found as a result of an uninformed search against the S. cerevisiae protein database. S_f, quality of match; TIC, total ion current; % TIC, signal or ion current for a given peptide; Sum TIC, sum of all TIC values for that protein identification—this exceeded the minimum required score in all cases. Additional information may be found at http://www.tucf.org. Complete LC–MS–MS scans are available upon request

Mss116p inhibits transcription by mtRNAP. (A) Effect of Mss116p on run-off transcription by Rpo41p. A double-stranded DNA template containing a consensus Rpo41p promoter (boxed, transcription start site is at + 1, A) was transcribed in the presence (+) or absence (−) of a five-fold molar excess of Mss116p over Rpo41p. Run-off products accumulated at each time point were resolved by electrophoresis in a 15% polyacrylamide gel and quantified by PhosphorImager analysis (shown on graph); arrow indicates position of expected 77 nt run-off product. (B) Dose-dependence of inhibition. Transcription was carried out in the presence of increasing amounts of Mss116p for 15 min, and the products were analysed as described above. Transcription activity is expressed relative to the level observed in the absence of Mss116p. Error bars represent standard error for run-off transcripts (n = 10). (C) Effects of exogenous RNA, DNA or protein. Transcription was carried out in the presence or absence of a five-fold molar excess of Mss116p for 15 min in the presence or absence of a 50-fold excess of non-specific analogues of transcription complex components (DNA, RNA, protein) or of an RNA with a secondary structure (tRNA), as noted. The level of inhibition by Mss116p in the presence of each competitor is indicated as a percentage of the activity observed in the absence of Mss116p (control, 100%). The presence of competitor alone had no effect on RNAP activity, as shown for tRNA in the right panel