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Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression.

Cotney J, Wang Z, Shadel GS - Nucleic Acids Res. (2007)

Bottom Line: Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression.Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis.Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, 440 Clifton Road N.E., Atlanta, Georgia 30322, USA.

ABSTRACT
Human mitochondrial transcription requires the bacteriophage-related RNA polymerase, POLRMT, the mtDNA-binding protein, h-mtTFA/TFAM, and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. Here, we determined the steady-state levels of these core transcription components and examined the consequences of purposeful elevation of h-mtTFB1 or h-mtTFB2 in HeLa cells. On a per molecule basis, we find an approximately 6-fold excess of POLRMT to mtDNA and approximately 3-fold more h-mtTFB2 than h-mtTFB1. We also estimate h-mtTFA at approximately 50 molecules/mtDNA, a ratio predicted to support robust transcription, but not to coat mtDNA. Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression. This is accompanied by increased translation rates of most, but not all mtDNA-encoded proteins. Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis. Furthermore, h-mtTFB1 mRNA and protein are elevated in response to h-mtTFB2 over-expression, suggesting the existence of a retrograde signal to the nucleus to coordinately regulate expression of these related factors. Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function.

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Analysis of mitochondrial translation rates and kasugamycin sensitivity in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. (A) Mitochondrial translation products were labeled in vivo with 35S-methionine in the presence of the cytoplasmic translation inhibitor emetine. Mitochondria were purified after labeling and 100 μg of mitochondrial protein were loaded on a linear gradient polyacrylamide gel. Shown on the left is a coomassie stain of the resulting gel demonstrating equal protein loading and on the right is an autoradiogram of the radiolabeled mitochondrial proteins (specific proteins are indicated on the right). Changes in the profile of labeled products only occur during over-expression of h-mtTFB2. (B) Plotted is the percent viable cells of the indicated cell lines after growth for 72 h in the indicated amount of the aminoglycoside kasugamycin. The analysis was done in triplicate and values shown are the mean ± SD.
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Figure 3: Analysis of mitochondrial translation rates and kasugamycin sensitivity in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. (A) Mitochondrial translation products were labeled in vivo with 35S-methionine in the presence of the cytoplasmic translation inhibitor emetine. Mitochondria were purified after labeling and 100 μg of mitochondrial protein were loaded on a linear gradient polyacrylamide gel. Shown on the left is a coomassie stain of the resulting gel demonstrating equal protein loading and on the right is an autoradiogram of the radiolabeled mitochondrial proteins (specific proteins are indicated on the right). Changes in the profile of labeled products only occur during over-expression of h-mtTFB2. (B) Plotted is the percent viable cells of the indicated cell lines after growth for 72 h in the indicated amount of the aminoglycoside kasugamycin. The analysis was done in triplicate and values shown are the mean ± SD.

Mentions: Both h-mtTFB1 and h-mtTFB2 have retained rRNA methyltransferase activity that is postulated to modulate mitochondrial ribosome biogenesis and/or translation (5). This, coupled to the fact that a function for Drosophila mtTFB1 in mitochondrial translation has been documented (19), we next measured mitochondrial translation rates in the h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines using an in vivo-radiolabeling approach. Similar to the analysis of the other mitochondrial gene expression parameters (Figure 2), we again saw major differences only in the h-mtTFB2 over-expression cell line, where a significant increase in the overall rate of translation was observed (Figure 3A). Interestingly, however, the labeling of specific products was not uniform (e.g. COX1, COX2 and ATP6), suggesting that increasing the amount of h-mtTFB2 and/or mitochondrial mRNAs is leading to increased translation of some mRNAs to the exclusion of others.Figure 3.


Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression.

Cotney J, Wang Z, Shadel GS - Nucleic Acids Res. (2007)

Analysis of mitochondrial translation rates and kasugamycin sensitivity in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. (A) Mitochondrial translation products were labeled in vivo with 35S-methionine in the presence of the cytoplasmic translation inhibitor emetine. Mitochondria were purified after labeling and 100 μg of mitochondrial protein were loaded on a linear gradient polyacrylamide gel. Shown on the left is a coomassie stain of the resulting gel demonstrating equal protein loading and on the right is an autoradiogram of the radiolabeled mitochondrial proteins (specific proteins are indicated on the right). Changes in the profile of labeled products only occur during over-expression of h-mtTFB2. (B) Plotted is the percent viable cells of the indicated cell lines after growth for 72 h in the indicated amount of the aminoglycoside kasugamycin. The analysis was done in triplicate and values shown are the mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1919481&req=5

Figure 3: Analysis of mitochondrial translation rates and kasugamycin sensitivity in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. (A) Mitochondrial translation products were labeled in vivo with 35S-methionine in the presence of the cytoplasmic translation inhibitor emetine. Mitochondria were purified after labeling and 100 μg of mitochondrial protein were loaded on a linear gradient polyacrylamide gel. Shown on the left is a coomassie stain of the resulting gel demonstrating equal protein loading and on the right is an autoradiogram of the radiolabeled mitochondrial proteins (specific proteins are indicated on the right). Changes in the profile of labeled products only occur during over-expression of h-mtTFB2. (B) Plotted is the percent viable cells of the indicated cell lines after growth for 72 h in the indicated amount of the aminoglycoside kasugamycin. The analysis was done in triplicate and values shown are the mean ± SD.
Mentions: Both h-mtTFB1 and h-mtTFB2 have retained rRNA methyltransferase activity that is postulated to modulate mitochondrial ribosome biogenesis and/or translation (5). This, coupled to the fact that a function for Drosophila mtTFB1 in mitochondrial translation has been documented (19), we next measured mitochondrial translation rates in the h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines using an in vivo-radiolabeling approach. Similar to the analysis of the other mitochondrial gene expression parameters (Figure 2), we again saw major differences only in the h-mtTFB2 over-expression cell line, where a significant increase in the overall rate of translation was observed (Figure 3A). Interestingly, however, the labeling of specific products was not uniform (e.g. COX1, COX2 and ATP6), suggesting that increasing the amount of h-mtTFB2 and/or mitochondrial mRNAs is leading to increased translation of some mRNAs to the exclusion of others.Figure 3.

Bottom Line: Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression.Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis.Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, 440 Clifton Road N.E., Atlanta, Georgia 30322, USA.

ABSTRACT
Human mitochondrial transcription requires the bacteriophage-related RNA polymerase, POLRMT, the mtDNA-binding protein, h-mtTFA/TFAM, and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. Here, we determined the steady-state levels of these core transcription components and examined the consequences of purposeful elevation of h-mtTFB1 or h-mtTFB2 in HeLa cells. On a per molecule basis, we find an approximately 6-fold excess of POLRMT to mtDNA and approximately 3-fold more h-mtTFB2 than h-mtTFB1. We also estimate h-mtTFA at approximately 50 molecules/mtDNA, a ratio predicted to support robust transcription, but not to coat mtDNA. Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression. This is accompanied by increased translation rates of most, but not all mtDNA-encoded proteins. Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis. Furthermore, h-mtTFB1 mRNA and protein are elevated in response to h-mtTFB2 over-expression, suggesting the existence of a retrograde signal to the nucleus to coordinately regulate expression of these related factors. Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function.

Show MeSH
Related in: MedlinePlus