Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.DNA-dependent RNA synthesis initiates with the specific binding of the RNA polymerase (RNAP) 2 to its promoter. During this process of promoter selection, the RNAP is able to seek out active promoters within vast stretches of non-promoter sequences in the genome. The multi-subunit RNAPs of the bacteria, archaea, and eukarya rely on transcription factors for promoter selection and specific transcription (1-3) whereas the single subunits RNAPs of bacteriophages are able to carry out the same functions without any accessory factors (4). The RNAPs that transcribe the mitochondrial genomes are unique in that they are homologous to the single subunit RNAPs of bacteriophages, but they require one or more transcription factors for promoter-specific initiation (5-9). The transcription factors modulate various steps of initiation and play a key role in open complex formation (10,11).In this study, we have investigated the transcription machinery of the yeast (Saccharomyces cerevisiae) mitochondria, which consists of a nuclear encoded ϳ153 kDa core subunit Rpo41 (12) and a ϳ45 kDa transcription factor Mtf1 (13, 14). The two proteins are sufficient to catalyze transcription from the conserved nona-nucleotide promoter (5Ј-ATATA-AGTA(ϩ1)) of the yeast mitochondria that directs the synthesis of rRNA, tRNA, and respiratory chain protein mRNAs (15). Rpo41 cannot initiate specific transcription on duplex promoters without Mtf1 (13,16,17). However, when the DNA is negatively supercoiled or premelted, then Rpo41 can initiate specific transcription without Mtf1 (18). Based on these results, it was proposed that Rpo41 has the intrinsic ability to recognize the promoter. A similar conclusion was made for its homolo...

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 ...

Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP “sensor” to regulate gene expression.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.