Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase

Wijngaert, Brent De; Sultana, Shemaila; Singh, Anupam; Dharia, Chhaya; Vanbuel, Hans; Shen, Jiayu; Vasilchuk, Daniel; Martinez, Sergio E.; Kandiah, Eaazhisai; Patel, Smita S.; Das, Kalyan

doi:10.1016/j.molcel.2020.11.016

Cited by 18 publications

(25 citation statements)

References 68 publications

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“…However, non-H-bonding mutations of these amino acids did not affect the transcription termination at embedded ribonucleotides. Interestingly, recent structural analysis of yeast mitochondrial RNA polymerase demonstrates that Tyr831, which is analogous to human Tyr807, interacts with the base and not with the sugar, which could explain the lack of an effect observed here ( 37 ). Presently, our working model is that the transcription termination observed here is due to structural changes in the template.…”

Section: Discussionmentioning

confidence: 64%

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression

Singh

Posse

Peter

et al. 2022

Nucleic Acids Research

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Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.

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Section: Discussionmentioning

confidence: 64%

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression

Singh

Posse

Peter

et al. 2022

Nucleic Acids Research

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“…These interactions likely occur after the initial promoter melting, as only the nucleotides in proximity to the TSS are recognized. Recognition of the non-template bases could be due to their flipping out and insertion into the pockets on the TFB2M surface, as observed in the yeast IC ( 33 ). Contrary to the SP loop's role in T7 RNAP (Cheetham et al, 1999), we detected only a moderate (10–50%) decrease in transcription efficiency when substitutions were introduced in the -6 to -12 promoter region.…”

Section: Discussionmentioning

confidence: 98%

Mechanisms of mitochondrial promoter recognition in humans and other mammalian species

Zamudio-Ochoa

Morozov

Sarfallah

et al. 2022

Nucleic Acids Research

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Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence-specific protein-DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species-specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.

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“…Prediction of the Rpo41 secondary structure was performed using the PSIPRED 4.0 Workbench (UCL-CS Bioinformatics, London, United Kingdom) ( Buchan and Jones, 2019 ). For homology modeling of the Rpo41 protein, the Hidden Markov Model-based tool HHPred ( Zimmermann et al, 2018 ) and MODELLER 9.25 ( Webb and Sali, 2016 ) were used, based on the highly similar crystal structure of yeast mitochondrial RNA polymerase from S. cerevisiae (Protein Data Bank, PDB 6YMW) with 98.94% probability and 1.4 × 10 –21 E -value, as described previously ( Deshpande and Patel, 2012 ; De Wijngaert et al, 2021 ). Protein structures were visualized and compared using PyMOL 2.4.…”

Section: Methodsmentioning

confidence: 99%

“…Regarding the most important proteins implicated in fungal mitochondrial transcription, two proteins, i.e., mtRNA polymerase (mtRNAP or Rpo41) and Mtf1 play the most significant roles ( Lipinski et al, 2010 ; De Wijngaert et al, 2021 ). Both proteins are encoded by nuclear genes, i.e., rpo 41 and mtf 1, as the former is the catalytic component of the transcription and the latter is the sole needed transcription factor for the orderly function of the polymerase, in contrast to the nuclear polymerases for which many different transcription factors are needed ( Yang et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial Transcription of Entomopathogenic Fungi Reveals Evolutionary Aspects of Mitogenomes

Varassas

Kouvelis

2022

Front. Microbiol.

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Entomopathogenic fungi and more specifically genera Beauveria and Metarhizium have been exploited for the biological control of pests. Genome analyses are important to understand better their mode of action and thus, improve their efficacy against their hosts. Until now, the sequences of their mitochondrial genomes were studied, but not at the level of transcription. Except of yeasts and Neurospora crassa, whose mt gene transcription is well described, in all other Ascomycota, i.e., Pezizomycotina, related information is extremely scarce. In this work, mt transcription and key enzymes of this function were studied. RT-PCR experiments and Northern hybridizations reveal the transcriptional map of the mt genomes of B. bassiana and M. brunneum species. The mt genes are transcribed in six main transcripts and undergo post-transcriptional modifications to create single gene transcripts. Promoters were determined in both mt genomes with a comparative in silico analysis, including all known information from other fungal mt genomes. The promoter consensus sequence is 5′-ATAGTTATTAT-3′ which is in accordance with the definition of the polycistronic transcripts determined with the experiments described above. Moreover, 5′-RACE experiments in the case of premature polycistronic transcript nad1-nad4-atp8-atp6 revealed the 5′ end of the RNA transcript immediately after the in silico determined promoter, as also found in other fungal species. Since several conserved elements were retrieved from these analyses compared to the already known data from yeasts and N. crassa, the phylogenetic analyses of mt RNA polymerase (Rpo41) and its transcriptional factor (Mtf1) were performed in order to define their evolution. As expected, it was found that fungal Rpo41 originate from the respective polymerase of T7/T3 phages, while the ancestor of Mtf1 is of alpha-proteobacterial origin. Therefore, this study presents insights about the fidelity of the mt single-subunit phage-like RNA polymerase during transcription, since the correct identification of mt promoters from Rpo41 requires an ortholog to bacterial sigma factor, i.e., Mtf1. Thus, a previously proposed hypothesis of a phage infected alpha-proteobacterium as the endosymbiotic progenitor of mitochondrion is confirmed in this study and further upgraded by the co-evolution of the bacterial (Mtf1) and viral (Rpo41) originated components in one functional unit.

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Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase

Cited by 18 publications

References 68 publications

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression

Mechanisms of mitochondrial promoter recognition in humans and other mammalian species

Mitochondrial Transcription of Entomopathogenic Fungi Reveals Evolutionary Aspects of Mitogenomes

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