The mitochondrial RNA polymerase (mtRNAP) of Saccharomyces cerevisiae, consisting of a complex of Rpo41 and Mtf1, is homologous to the phage single polypeptide T7/T3 RNA polymerases. The yeast mtRNAP recognizes a conserved nonanucleotide sequence to initiate specific transcription. In this work, we have defined the region of the nonanucleotide that is melted by the mtRNAP using 2-aminopurine (2AP) fluorescence that is sensitive to changes in base stacking interactions. We show that mtRNAP spontaneously melts the promoter from ؊4 to ؉2 forming a bubble around the transcription start site at ؉1. The location and size of the DNA bubble in this open complex of the mtRNAP closely resembles that of the T7 RNA polymerase. We show that DNA melting requires the simultaneous presence of Rpo41 and Mtf1. Adding the initiating nucleotide ATP does not expand the size of the initially melted DNA, but the initiating nucleotide differentially affects base stacking interactions at ؊1 and ؊2. Thus, the promoter structure upstream of the transcription start site is slightly rearranged during early initiation from its structure in the pre-initiation stage. Unlike on the duplex promoter, Rpo41 alone was able to form a competent open complex on a pre-melted promoter. The results indicate that Rpo41 contains the elements for recognizing the melted promoter through interactions with the template strand. We propose that Mtf1 plays a role in base pair disruption during the early stages of open complex formation.The mitochondrial genome is transcribed by a distinct RNA polymerase, which is simpler in composition when compared with the nuclear RNA polymerases that transcribe the cellular genome. The mitochondrial RNA polymerases (mtRNAP) 2 are highly homologous to the single subunit bacteriophage T7/T3 RNA polymerase (1-4). The transcription machinery of the yeast (Saccharomyces cerevisiae) mitochondria is comprised of a core polymerase, Rpo41, encoded by nuclear RPO41 gene and a transcription factor, Mtf1 (also referred as sc-mtTFB), encoded by nuclear MTF1 gene (1, 5, 6). Both proteins are transported into the mitochondria where they form a heterodimeric Rpo41/Mtf1 complex (mtRNAP) (7). Recent studies have shown that in humans, an alternative transcript of the mtRNAP gene localizes in the nucleus and the truncated protein is responsible for the transcription of select nuclear mRNAs (8).Unlike the single subunit RNAP of bacteriophages that do not require accessory proteins for transcription initiation, accessory factors are required to initiate specific transcription at the mtRNAP promoters. In yeast, Mtf1 is required in addition to Rpo41 for sequence-specific transcription on linear mtDNA templates (9 -11). While the crystal structure of Mtf1 shows unexpected resemblance to ErmCЈ RNA methyltransferase from Bacillus subtilis (12), there is no documented rRNA methyltransferase activity for Mtf1. In mammalian and insect systems, mitochondrial transcription in vitro requires at least three core components including the core RNA polymerase, mt...
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...
Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ϳ80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ϳ50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1-270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1-380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.The mitochondrial (mt) 2 genome of the yeast (Saccharomyces cerevisiae) is transcribed by nuclear DNA-encoded RNA polymerase (RNAP) subunits that produce ribosomal RNAs, transfer RNA, and mRNAs of the proteins of the oxidative phosphorylation machinery (1). The yeast transcription machinery consists of the core RNAP subunit, a 153-kDa protein called Rpo41, and the transcription factor, a 40-kDa protein called Mtf1 (2-5). Rpo41 is evolutionarily related to the single-subunit phage T7/T3 RNAP (6). Although the ϳ80-kDa C-terminal domain of Rpo41 is highly homologous to the phage T7/T3 RNAPs and conserved across the mitochondrial RNAPs of yeasts, plants, animals, and humans, the ϳ50-kDa N-terminal domain (NTD) is only conserved in few yeasts and absent in single subunit T7/T3 RNAPs (supplemental Table 1) (6, 7).The yeast Rpo41 NTD has been implicated previously to play a role in RNA processing and translation (8). However, whether the NTD of the Rpo41 plays a role in transcription related functions has not been determined. Deletion of 1-200 amino acids (aa) causes temperature sensitive petite phenotype in S. cerevisiae and results in mitochondrial genome instability, whereas deletions beyond 200 aa result in the RPO41 null phenotype (9). Subsequently, it was shown that the region 118 -208 NTD of the Rpo41 harbors a binding site for the Nam1p protein, which is involved in ...
We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.