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 promoterspecific 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. The mitochondrial (mt)2 RNA polymerase (RNAP) is closely related to the single-subunit bacteriophage T7/T3 RNAP (1). Their C-terminal ϳ800 amino acids show ϳ30% sequence identity to T7 RNAP (1-3). Despite this similarity, the mtRNAP depends on transcription factors for promoter-specific initiation (4 -7). The core RNAP subunit of the Saccharomyces cerevisiae, Rpo41, requires one major factor, Mtf1 (or sc-MtfB) (6, 8 -11), whereas the human core mtRNAP requires two factors, mtTFB1/2 and mtTFA (12-17).Rpo41 by itself does not recognize and melt the mt promoter or initiate RNA synthesis unless the promoter is premelted around the transcription site (18 -20). Similarly, there is no evidence that Mtf1 by itself interacts with the promoter (7, 18). When Mtf1 and Rpo41 are present together, the complex (21) melts the promoter from Ϫ4 to ϩ2 without requiring initiating NTPs (18). Based on these results, it has been suggested that Rpo41 lacks the mechanism for melting/stabilizing the open promoter and relies on Mtf1 for promoter-specific binding, melting, and stabilizing of the melted promoter.There are two general ways in which Mtf1 can facilitate promoter opening (Fig. 1). In Model A, only the Rpo41 protein within the mtRNAP complex (Rpo41⅐Mtf1) interacts with the promoter DNA, and Mtf1 acts allosterically. Through protein-protein interactions, Mtf1 causes conformational changes with...

The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

“…The direction of transcription (arrow) from the start site (ϩ1) and the conserved nonanucleotide sequence from Ϫ8 to ϩ1 (underlined) are shown. B, FL Rpo41 or the NTD mutant (2 M), Mtf1 (2.5 M), and U25D32ds (2.5 M) were reacted with 250 M ATP (ϩ␥-32 PATP), UTP, and GTP at 25°C for 30, 60, 120, 180, and 300 s. The 23% polyacrylamide-urea sequencing gel image shows the RNA products from the transcription reactions with FL Rpo41-Mtf1 (lanes 1-5), DN100-Mtf1(lanes 6 -10), DN270-Mtf1 (lanes [11][12][13][14][15], and DN380-Mtf1 (lanes 16 -20). Lane 21 shows a control reaction containing Mtf1, DNA and ATP, UTP, and GTP but without Rpo41.…”

“…Fluorescencebased studies have shown that the Rpo41-Mtf1 complex melts the promoter from Ϫ4 to ϩ2, but Rpo41 alone does not melt the promoter (13). Recent cross-linking studies indicated that Mtf1 interacts with the promoter DNA via its C-terminal amino acids, and these interactions might aid in promoter opening (14,15). Earlier studies have shown that although Mtf1 is present in the initiation complex with Rpo41, it is released after 13-mer RNA synthesis (16).…”

“…Based on previously reported studies (9,12), we hypothesized that the deletion of 100 aa from the NTD should minimally affect the transcription activity of Rpo41, but deletions larger than ϳ200 aa would have significant effects on the transcription activity. Deletion of 380 creates a minimal catalytic domain of Rpo41, within 100 aa of the predicted sequence similarity between Rpo41 and T7 RNAP (14), and therefore, its characterization was particularly interesting. Our detailed biochemical investigation of DN100, DN270, and DN380 proteins show that the NTD of Rpo41 is autoinhibitory to its transcription activity.…”

The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding

“…[9][10][11] In contrast to phage RNAPs, mitochondrial RNAPs require accessory factors for promoter recognition, melting and termination. 12,13 T7 DNA and RNA polymerases achieve complex stability while allowing lateral movement via their thumb subdomains. In T7DNAP, an extended loop of the thumb subdomain interacts with thioredoxin to present a large surface that interacts with DNA, and in T7RNAP the thumb subdomain is a flexible element important for processivity, stability of the late initiation and elongation complexes, and recognition of Class II termination sequences.…”

Pentatricopeptide motifs in the N-terminal extension domain of yeast mitochondrial RNA polymerase Rpo41p are not essential for its function

“…It is a complex composed of the 153 kDa catalytic subunit encoded by the RPO41 gene, and a single accessory 40 kDa transcription factor, encoded by the MTF1 gene [7][8][9][10][11][12]. Whereas Rpo41p can recognize and initiate transcription from premelted promoter sequences in vitro [13], Mtf1p is required for transcription initiation from duplex DNA [14,15]. The Rpo41p-Mtf1p complex recognises the promoter sequence by a mechanism reliant on induced fit and DNA bending [14].…”