The core human mitochondrial transcription initiation complex

Shutt, Timothy E.; Bestwick, Megan; Shadel, Gerald S.

doi:10.4161/trns.2.2.14296

Cited by 47 publications

(50 citation statements)

References 30 publications

(44 reference statements)

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“…Within the limits of resolution of this approach, which relies on confocal microscopy, and not on super-resolution microscopy (Brown et al, 2011;Kukat et al, 2011), labelling of mtDNAs and RNAs was also shown to be correlated with nucleoids, the mitochondrial substructures involved in mtDNA processing. We observed different levels of colocalization between FISH and nucleoid markers, in agreement with the different amounts of regulatory proteins found in nucleoids and which might have regulatory functions (Chen and Butow, 2005;Shutt et al, 2011;Spelbrink, 2010). mTRIP detects mitochondria and mitochondrial substructures rich in a given transcript.…”

Section: Discussionsupporting

confidence: 60%

Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging

Châtre

Ricchetti

2012

Journal of Cell Science

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SummaryMitochondrial DNA (mtDNA) replication and transcription are crucial for cell function, but these processes are poorly understood at the single-cell level. We describe a novel fluorescence in situ hybridization protocol, called mTRIP (mitochondrial transcription and replication imaging protocol), that reveals simultaneously mtDNA and RNA, and that can also be coupled to immunofluorescence for in situ protein examination. mTRIP reveals mitochondrial structures engaged in initiation of DNA replication by identification of a specific sequence in the regulatory D-loop, as well as unique transcription profiles in single human cells. We observe and quantify at least three classes of mitochondrial structures: (i) replication initiation active and transcript-positive (Ia-Tp); (ii) replication initiation silent and transcript-positive (Is-Tp); and (iii) replication initiation silent and transcript-negative (Is-Tn). Thus, individual mitochondria are dramatically heterogeneous within the same cell. Moreover, mTRIP exposes a mosaic of distinct nucleic acid patterns in the D-loop, including H-strand versus L-strand transcripts, and uncoupled rRNA transcription and mtDNA initiation of replication, which might have functional consequences in the regulation of the mtDNA. Finally, mTRIP identifies altered mtDNA processing in cells with unbalanced mtDNA content and function, including in human mitochondrial disorders. Thus, mTRIP reveals qualitative and quantitative alterations that provide additional tools for elucidating the dynamics of mtDNA processing in single cells and mitochondrial dysfunction in diseases.

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Section: Discussionsupporting

confidence: 60%

Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging

Châtre

Ricchetti

2012

Journal of Cell Science

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“…TFAM had initially been classified as a core transcription factor (23), but more recent evidence suggests a similarly biphasic role in the modulation of HSP1 and LSP, with activation and repression predominant at different TFAM concentrations (9). This finding has given rise to the suggestion that TFAM acts to modulate mitochondrial transcription, increasing the availability of rRNAs at low concentration, increasing mRNA and mtDNA replication at moderate concentration, and shutting both transcription and replication down at high concentration (24,25).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

Zollo

Tiranti

Sondheimer

2012

Proc. Natl. Acad. Sci. U.S.A.

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The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis.mitochondrial biology | organelle T he mitochondrial genome (mtDNA) encodes 13 proteins that are constituents of the electron transport chain and ATP synthase. Transcription of mtDNA proceeds bidirectionally, radiating out from the noncoding D-loop. The light-strand promoter (LSP) is required for the synthesis of ND6 mRNA and eight of the mitochondrial tRNAs (1). Transcription from LSP also primes the origin of heavy-strand replication, which is the first step in the synthesis of mtDNA (2).The heavy strand encodes 12 mRNAs, both mitochondrial rRNAs and the remaining tRNAs. Intracellular levels of mature rRNAs are in great excess to the mRNAs (3). Although it was initially proposed that this imbalance was caused by the existence of a single initiation site, with most primary transcripts terminating before reaching the protein coding region, later studies used a combination of ribonucleotide incorporation assays and capping of precursor transcripts to suggest the existence of two distinct heavy-strand initiation sites in close proximity (4-6). The first heavy-strand promoter (HSP1), which lies fully within the Dloop, is responsible for the synthesis of a transcript composed of the 12S and 16S rRNAs and intervening tRNAs (Fig. 1A). The minor or distal heavy-strand promoter (HSP2) initiates within the sequence of the mitochondrial tRNA for phenylalanine. HSP2-initiated transcription produces the remaining mitochondrial mRNAs by processing nearly the full length of the mtDNA.Studies of HSP2 promoter function are complicated by its unusual position, both within a tRNA-...

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“…Such study highlights the possibility that h-TFAM, by virtue of its abundance or activity, could dictate relative mtDNA promoter utilization via differential transcriptional activation of HSP1 and LSP. Given that transcription from the LSP is involved in priming of H-strand mtDNA replication and is exquisitely sensitive to the levels of h-TFAM, it was speculated that individual molecules or nucleoids (complexes estimated to contain 2-10 mtDNA molecules) that have different amounts of h-TFAM bound could have different transcription and replication potential (Shutt et al, 2011). Therefore, it has been concluded that the core human mitochondrial transcription complex is actually a two-component system orthologous and homologous to that of yeast and that h-TFAM acts as a transcriptional activator instead of being an obligate core component of the transcription initiation complex (Shutt et al, 2011).…”

Section: Tfam In Transcriptionmentioning

confidence: 99%

“…Given that transcription from the LSP is involved in priming of H-strand mtDNA replication and is exquisitely sensitive to the levels of h-TFAM, it was speculated that individual molecules or nucleoids (complexes estimated to contain 2-10 mtDNA molecules) that have different amounts of h-TFAM bound could have different transcription and replication potential (Shutt et al, 2011). Therefore, it has been concluded that the core human mitochondrial transcription complex is actually a two-component system orthologous and homologous to that of yeast and that h-TFAM acts as a transcriptional activator instead of being an obligate core component of the transcription initiation complex (Shutt et al, 2011). This is conceptually appealing, but such hypothetical scenario requires that the amount (and/or activity) of h-TFAM in individual nucleoids (or perhaps even individual mtDNA molecules) is regulated in some manner.…”

Section: Tfam In Transcriptionmentioning

confidence: 99%

Mitochondrial transcription factor A (TFAM): one actor for different roles

Lezza

2012

Front. Biol.

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Mitochondrial Transcription Factor A (TFAM) is a nuclear-encoded factor present in mitochondria from all kinds of animals, even with a homologous form in yeast, which has strongly and progressively gained an eminent relevance in the regulation of mitochondrial DNA (mtDNA) functions. This is due to the various functions performed by the protein, which cover a very wide scenario including: replication, transcription, maintenance and eventually repair of mtDNA molecules. In consideration of so many different roles it plays, it can be understood there is a deep interest in its regulation of expression and activity as well as its eventual involvement in pathologies related to a decreased functionality/presence of the mitochondrial genome and the consequences of TFAM induced overexpression.

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The core human mitochondrial transcription initiation complex

Cited by 47 publications

References 30 publications

Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging

Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

Mitochondrial transcription factor A (TFAM): one actor for different roles

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