Mitochondrial transcription factor Mtf1 traps the unwound non-template strand to facilitate open complex formation

Abstract

The catalytic subunit of the mitochondrial (mt) RNA polymerase (RNAP) is highly homologous to the bacteriophage T7/T3 RNAP. Unlike the phage RNAP, however, the mtRNAP relies on accessory proteins to initiate promoter-specific transcription. Rpo41, the catalytic subunit of the Saccharomyces cerevisiae mtRNAP, requires Mtf1 for opening the duplex promoter. To elucidate the role of Mtf1 in promoter-specific DNA opening, we have mapped the structural organization of the mtRNAP using site-specific protein-DNA photo-cross-linking studies. Both Mtf1 and Rpo41 cross-linked to distinct sites on the promoter DNA, but the dominant cross-links were those of the Mtf1, which indicates a direct role of Mtf1 in promoter-specific binding and initiation. Strikingly, Mtf1 cross-linked with a high efficiency to the melted region of the promoter DNA, based on which we suggest that Mtf1 facilitates DNA melting by trapping the non-template strand in the unwound conformation. Additional strong cross-links of the Mtf1 were observed with the -8 to -10 base-paired region of the promoter. The cross-linking results were incorporated into a structural model of the mtRNAP-DNA, created from a homology model of the C-terminal domain of Rpo41 and the available structure of Mtf1. The promoter DNA is sandwiched between Mtf1 and Rpo41 in the structural model, and Mtf1 closely associates mainly with one face of the promoter across the entire nona-nucleotide consensus sequence. Overall, the studies reveal that in many ways the role of Mtf1 is analogous to the transcription factors of the multisubunit RNAPs, which provides an intriguing link between single- and multisubunit RNAPs.

FIGURE 1.

Proposed models of Mtf1 mechanism in promoter melting. Two models of promoter melting by Mtf1 are shown. Rpo41 (gray) and Mtf1 (brown-gray) can assemble in the absence of promoter DNA to form a complex (mtRNAP) (21). In the presence of promoter DNA (the red strand denotes the non-template strand, and the green strand denotes the template strand) two scenarios can be visualized. In model A, Mtf1 interacts only with Rpo41; binding of Mtf1 to Rpo41 causes a change in the conformation of Rpo41 and/or Mtf1 to facilitate DNA melting, where the DNA is melted from −4 to +2. In Model B, Mtf1 directly interacts with the promoter DNA and/or Rpo41 to facilitate promoter melting.

FIGURE 2.

Photo-cross-linking of Rpo41·Mtf1 in the initial open complex and initial transcribing complex with ATP. a, a 45-bp DNA with the 14 S rRNA promoter consensus promoter (in bold) was used in the cross-linking studies. The melted region in the initial open complex is highlighted in gray, and the arrows indicate the sites of derivatization. The nomenclature NT(−8/−9) indicates that the backbone phosphate between positions −8 and −9 on the NT was phosphorothioated and derivatized with 4-azidophenacylbromide. The nomenclature T(−8/−9) indicates a DNA in which the complementary position of NT(−8/−9) on the T was phosphorothioated and derivatized with 4-azido-phenacylbromide. b, cross-linking reactions were carried out with an equimolar mixture of Rpo41 and Mtf1 (100 nm each) and 300 nm radiolabeled DNA (derivatized at a single defined site) in the absence of ATP (−ATP panel) or in the presence of 1 mm ATP (+ATP panel). Cross-linked protein-DNA complexes were resolved on a 4–15% SDS-PAGE gel. Protein-DNA cross-linked products of Rpo41·Mtf1 complex with each of the derivatized DNAs on the non-template (NT) strand (lanes 1–12). Lanes CR and CM are control reactions with Rpo41 alone on NT(−5/−6) or Mtf1 alone on (NT +1/+2), respectively. The CE of Rpo41 and Mtf1 was determined using Equation 1 and plotted using the same y scale for better comparison. Error bars were calculated from three independent experiments. c, protein-DNA cross-linked products of Rpo41·Mtf1 to the T strand (lanes 1–12). The calculations are similar to those of the NT strand. Dashed lines within c (−ATP and +ATP panels) indicate the incorporation of additional lanes.

FIGURE 3.

Photo-cross-linking of Rpo41·Mtf1 with the pre-melted promoters. a, the sequence of the pre-melted promoter is shown with a non-consensus NT sequence from −4 to +2 to create mismatches in that region. The arrow indicates the location and direction of transcription initiation. b, shown is RNA synthesis from derivatized pre-melted DNAs. The suffix B (bubble) after each position indicates derivatization of that position in the pre-melted DNA. 500 nm Rpo41 (lanes 2, 4, and 6) or an equimolar mixture of Rpo41 and Mtf1 (500 nm each) (lanes 1, 3, and 5) were mixed with derivatized pre-melted DNA (1 μm), ATP, UTP, and GTP (250 μm each) spiked with [γ-³²P]ATP for 3 min at 22 °C. The RNA products were resolved on an 18% polyacrylamide sequencing gel containing 7 m urea. The first C base in the DNA sequence is encountered at +21. Exclusion of CTP, therefore, results in a runoff product of 20 nt. c, a 4–15% PAGE gel show the results of the cross-linking reaction with each of the derivatized DNAs. The cross-linking efficiencies for duplex DNA and pre-melted DNA are shown for comparison.

FIGURE 4.

A structural model of the S. cerevisiae mitochondrial RNAP open complex. a, a summary of the protein-DNA cross-linking results is shown. The purple spheres represent Mtf1 cross-links, and the yellow ones represent those of the Rpo41 (Fig. 2). The size of the sphere is proportional to the CE (supplemental Fig. S7). b, a structural model of the mtRNAP open complex is shown. The Rpo41 (416–1214) structure is based on homology modeling to T7 RNAP (PDB code 1QLN) (supplemental information). This region of Rpo41 represents just over half of the protein and shows the highest sequence similarity to T7 RNAP. The DNA is in the same conformation as in 1QLN. The non-template strand (NT) is shown in red, and the template strand (T) is in green. The orientation of Mtf1 is speculative. The placement of Mtf1 (PDB code 1I4W, shown in purple) is based on the cross-linking results from our studies and the proximity of the C-terminal tail of Mtf1 (in dark blue) to the promoter (42). The model shows the promoter sandwiched between Rpo41 and Mtf1 and Mtf1 contacting mainly one face of the promoter at −8/−10, the unwound NT strand, and the transcription start site region. c, shown is comparison of the T7 RNAP structure with the homology model of Rpo41. The three-dimensional structure of Rpo41, 416–1214 (in gray) is aligned with the three-dimensional structure of T7 RNAP, 6–883 residues (PDB code 1QLN) (light blue). The superimposition highlights the model-predicted DNA binding structural elements in Rpo41 that are homologous to the structural elements of T7 RNAP, namely the AT-rich recognition loop (yellow), the specificity loop (brown), and the intercalating hairpin (pink).