Fluorescence mapping of the open complex of yeast mitochondrial RNA polymerase.

Tang, Guo Qing; Paratkar, Swaroopa; Patel, Smita S.

doi:10.1074/jbc.a807880200

Cited by 6 publications

(19 citation statements)

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“…The N-terminal His-tagged POLRMT, N-terminal Histagged Rpo41, untagged TEFM, and untagged T7 RNAP were purified as described previously (25,28,36,44,57,58). Enzyme concentration was calculated from its absorbance at 280 nm and the calculated molar extinction coefficient.…”

Section: Nucleic Acids Proteins and Other Reagentsmentioning

confidence: 99%

“…Mitochondrial RNAPs are related to T7 RNAP (16 -18), but they depend on accessory factors for transcription initiation (19 -24). For example, the human POLRMT requires TFAM and TFB2M (25,26) for promoter opening, and the yeast Rpo41 requires Mtf1 (27,28). T7 RNAP is widely used in in vitro transcription reactions for RNA synthesis, and mitochondrial RNAPs play a key role in cellular energy production.…”

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confidence: 99%

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Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

Sultana¹,

Solotchi²,

Ramachandran³

et al. 2017

Journal of Biological Chemistry

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Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10), yeast mitochondrial Rpo41 (6 × 10), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10 The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine-purine mismatches were relatively frequent (10-10), whereas C→G, U→A, G→C, and C→A errors from purine-purine and pyrimidine-pyrimidine mismatches were rare (10-10). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of transcribed RNAs.

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Section: Nucleic Acids Proteins and Other Reagentsmentioning

confidence: 99%

mentioning

confidence: 99%

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

Sultana¹,

Solotchi²,

Ramachandran³

et al. 2017

Journal of Biological Chemistry

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“…Mitochondrial transcription factors, Mtf1 and TFB2M, of the yeast (S. cerevisiae) and human, respectively, have a well-established role in promoter melting during transcription initiation (8,11,17,20). These homologous transcription factors bind to the nontemplate strand of the transcription bubble to drive the promoter melting step.…”

Section: Discussionmentioning

confidence: 99%

“…1B). This DNA included the -4 to +2 region that forms a transcription bubble (17) and interacts with Mtf1 in the initiation complex (11).…”

Section: The C-tail Inhibits the Dna Binding Activity Of Mtf1mentioning

confidence: 99%

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding

Basu

Mishra

Farooqui

et al. 2020

Journal of Biological Chemistry

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The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

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“…Mtf1 on its own does not bind DNA because the C-terminal tail autoinhibits DNA binding 37 , but in complex with Rpo41, Mtf1 drives promoter melting by trapping the non-template strand of the initiation bubble at positions −4 to +2 (refs. 26,27,38,39 ). Biochemical and recent structural studies of yeast mitochondrial RNAP show that the promoter DNA is severely bent around the transcription start site in the open complex 39,40 .…”

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confidence: 99%

The dynamic landscape of transcription initiation in yeast mitochondria

et al. 2020

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Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use singlemolecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.

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Fluorescence mapping of the open complex of yeast mitochondrial RNA polymerase.

Cited by 6 publications

References 0 publications

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding

The dynamic landscape of transcription initiation in yeast mitochondria

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