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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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h-mtTFB2 is processed in vivo and its over-expression in HeLa cells results in a coordinated increase of h-mtTFB1, but not POLRMT or h-mtTFA. (A) Shown are western blots on 200 ng of recombinant h-mtTFB1 and h-mtTFB2 proteins probed with four peptide antibodies: TFB1-1, TFB1-2, TFB2-1 and TFB2-2. Antibodies were found to specifically recognize their full-length recombinant peptide and not cross react with the paralogous protein. Coomassie staining of full-length recombinant h-mtTFB1 and h-mtTFB2 (top panel) demonstrates loading and their difference in molecular weight. (B) Western blot of mitochondrial extracts (100 μg protein) from HeLa cell lines over-expressing h-mtTFB1 or h-mtTFB2 used in this study in parallel with recombinant h-mtTFB1 and h-mtTFB2 run as controls. The blot was probed as indicated using peptide antibodies that distinguish h-mtTFB1 and h-mtTFB2 (α-h-mtTFB1 and α-h-mtTFB2) and an antibody that recognizes HSP60 (α-HSP60) that was used as a mitochondrial loading control. The lanes are loaded as follows: lane 1, molecular weight markers; lane 2, recombinant h-mtTFB1; lanes 3, recombinant h-mtTFB2; lanes 4–8, mitochondrial extracts from an empty pcDNA 3.1 zeo (+) vector-control, h-mtTFB1 over-expression, and three different h-mtTFB2 stable over-expression HeLa cell lines, respectively. (C) Shown are the results of real-time RT-PCR measurement of h-mtTFB1 mRNA (see Supplementary Figure S3) in h-mtTFB1 and h-mtTFB2 over-expression cell lines relative to empty-vector control cells. Reverse transcriptase real-time PCR was used to measure Ct values for cDNA samples from listed cell lines. The Ct values for h-mtTFB1 mRNA were normalized to those of β-actin and the values shown were normalized to the ratio obtained in the empty-vector control, which was given a value of 1. Values shown are the mean ± SD for three separate measurements. (D) Western blot of mitochondrial lysates from the same cell lines described in B probed using antibodies that recognize h-mtTFA (α-h-mtTFA), POLRMT (α-POLRMT), and the outer mitochondrial membrane protein VDAC (α-VDAC/porin) as a mitochondrial loading control.
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Figure 1: h-mtTFB2 is processed in vivo and its over-expression in HeLa cells results in a coordinated increase of h-mtTFB1, but not POLRMT or h-mtTFA. (A) Shown are western blots on 200 ng of recombinant h-mtTFB1 and h-mtTFB2 proteins probed with four peptide antibodies: TFB1-1, TFB1-2, TFB2-1 and TFB2-2. Antibodies were found to specifically recognize their full-length recombinant peptide and not cross react with the paralogous protein. Coomassie staining of full-length recombinant h-mtTFB1 and h-mtTFB2 (top panel) demonstrates loading and their difference in molecular weight. (B) Western blot of mitochondrial extracts (100 μg protein) from HeLa cell lines over-expressing h-mtTFB1 or h-mtTFB2 used in this study in parallel with recombinant h-mtTFB1 and h-mtTFB2 run as controls. The blot was probed as indicated using peptide antibodies that distinguish h-mtTFB1 and h-mtTFB2 (α-h-mtTFB1 and α-h-mtTFB2) and an antibody that recognizes HSP60 (α-HSP60) that was used as a mitochondrial loading control. The lanes are loaded as follows: lane 1, molecular weight markers; lane 2, recombinant h-mtTFB1; lanes 3, recombinant h-mtTFB2; lanes 4–8, mitochondrial extracts from an empty pcDNA 3.1 zeo (+) vector-control, h-mtTFB1 over-expression, and three different h-mtTFB2 stable over-expression HeLa cell lines, respectively. (C) Shown are the results of real-time RT-PCR measurement of h-mtTFB1 mRNA (see Supplementary Figure S3) in h-mtTFB1 and h-mtTFB2 over-expression cell lines relative to empty-vector control cells. Reverse transcriptase real-time PCR was used to measure Ct values for cDNA samples from listed cell lines. The Ct values for h-mtTFB1 mRNA were normalized to those of β-actin and the values shown were normalized to the ratio obtained in the empty-vector control, which was given a value of 1. Values shown are the mean ± SD for three separate measurements. (D) Western blot of mitochondrial lysates from the same cell lines described in B probed using antibodies that recognize h-mtTFA (α-h-mtTFA), POLRMT (α-POLRMT), and the outer mitochondrial membrane protein VDAC (α-VDAC/porin) as a mitochondrial loading control.

Mentions: To fully understand any transcription system, a critical parameter to know is the relative amounts of the basal transcription machinery in vivo. Our approach was to use immunoblotting to establish the levels of the mitochondrial transcription machinery on a total cell and mitochondrial basis. This method requires antibodies that are specific for their target, especially for homologous proteins that are very similar in size. Since h-mtTFB1 and h-mtTFB2 share a large degree of sequence similarity and are predicted to migrate similarly on polyacrylamide gels, it was unclear if antibodies we had generated would cross react with the paralogous protein and confound our analysis. Therefore, we first generated peptide antibodies against h-mtTFB1 and h-mtTFB2 (see Materials and Methods section) and determined their specificity by assessing reactivity toward purified full-length recombinant h-mtTFB1 and h-mtTFB2 by immunoblotting. We found that these antibodies were indeed highly specific for their targets showing no reactivity with the paralogous protein (Figure 1A). Having antibodies capable of specifically detecting each of the four human mitochondrial transcription proteins, as well as known amounts of corresponding recombinant proteins, allowed us to determine for the first time their relative abundance. We purified mitochondria from logarithmically growing HeLa cells and performed quantitative western blot analysis of POLRMT, h-mtTFA, h-mtTFB1 and h-mtTFB2 on known amounts of total mitochondrial protein. For each protein analyzed, multiple dilutions of total mitochondrial protein or total whole-cell lysate were probed in parallel with a dilution series of known amounts of recombinant protein to allow the amount of each transcription component relative to the total amount of mitochondrial lysate to be determined. Representative western blots and standard curves showing linearity of the assays employed are shown in Supplementary Figure S1.Figure 1.


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)

h-mtTFB2 is processed in vivo and its over-expression in HeLa cells results in a coordinated increase of h-mtTFB1, but not POLRMT or h-mtTFA. (A) Shown are western blots on 200 ng of recombinant h-mtTFB1 and h-mtTFB2 proteins probed with four peptide antibodies: TFB1-1, TFB1-2, TFB2-1 and TFB2-2. Antibodies were found to specifically recognize their full-length recombinant peptide and not cross react with the paralogous protein. Coomassie staining of full-length recombinant h-mtTFB1 and h-mtTFB2 (top panel) demonstrates loading and their difference in molecular weight. (B) Western blot of mitochondrial extracts (100 μg protein) from HeLa cell lines over-expressing h-mtTFB1 or h-mtTFB2 used in this study in parallel with recombinant h-mtTFB1 and h-mtTFB2 run as controls. The blot was probed as indicated using peptide antibodies that distinguish h-mtTFB1 and h-mtTFB2 (α-h-mtTFB1 and α-h-mtTFB2) and an antibody that recognizes HSP60 (α-HSP60) that was used as a mitochondrial loading control. The lanes are loaded as follows: lane 1, molecular weight markers; lane 2, recombinant h-mtTFB1; lanes 3, recombinant h-mtTFB2; lanes 4–8, mitochondrial extracts from an empty pcDNA 3.1 zeo (+) vector-control, h-mtTFB1 over-expression, and three different h-mtTFB2 stable over-expression HeLa cell lines, respectively. (C) Shown are the results of real-time RT-PCR measurement of h-mtTFB1 mRNA (see Supplementary Figure S3) in h-mtTFB1 and h-mtTFB2 over-expression cell lines relative to empty-vector control cells. Reverse transcriptase real-time PCR was used to measure Ct values for cDNA samples from listed cell lines. The Ct values for h-mtTFB1 mRNA were normalized to those of β-actin and the values shown were normalized to the ratio obtained in the empty-vector control, which was given a value of 1. Values shown are the mean ± SD for three separate measurements. (D) Western blot of mitochondrial lysates from the same cell lines described in B probed using antibodies that recognize h-mtTFA (α-h-mtTFA), POLRMT (α-POLRMT), and the outer mitochondrial membrane protein VDAC (α-VDAC/porin) as a mitochondrial loading control.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: h-mtTFB2 is processed in vivo and its over-expression in HeLa cells results in a coordinated increase of h-mtTFB1, but not POLRMT or h-mtTFA. (A) Shown are western blots on 200 ng of recombinant h-mtTFB1 and h-mtTFB2 proteins probed with four peptide antibodies: TFB1-1, TFB1-2, TFB2-1 and TFB2-2. Antibodies were found to specifically recognize their full-length recombinant peptide and not cross react with the paralogous protein. Coomassie staining of full-length recombinant h-mtTFB1 and h-mtTFB2 (top panel) demonstrates loading and their difference in molecular weight. (B) Western blot of mitochondrial extracts (100 μg protein) from HeLa cell lines over-expressing h-mtTFB1 or h-mtTFB2 used in this study in parallel with recombinant h-mtTFB1 and h-mtTFB2 run as controls. The blot was probed as indicated using peptide antibodies that distinguish h-mtTFB1 and h-mtTFB2 (α-h-mtTFB1 and α-h-mtTFB2) and an antibody that recognizes HSP60 (α-HSP60) that was used as a mitochondrial loading control. The lanes are loaded as follows: lane 1, molecular weight markers; lane 2, recombinant h-mtTFB1; lanes 3, recombinant h-mtTFB2; lanes 4–8, mitochondrial extracts from an empty pcDNA 3.1 zeo (+) vector-control, h-mtTFB1 over-expression, and three different h-mtTFB2 stable over-expression HeLa cell lines, respectively. (C) Shown are the results of real-time RT-PCR measurement of h-mtTFB1 mRNA (see Supplementary Figure S3) in h-mtTFB1 and h-mtTFB2 over-expression cell lines relative to empty-vector control cells. Reverse transcriptase real-time PCR was used to measure Ct values for cDNA samples from listed cell lines. The Ct values for h-mtTFB1 mRNA were normalized to those of β-actin and the values shown were normalized to the ratio obtained in the empty-vector control, which was given a value of 1. Values shown are the mean ± SD for three separate measurements. (D) Western blot of mitochondrial lysates from the same cell lines described in B probed using antibodies that recognize h-mtTFA (α-h-mtTFA), POLRMT (α-POLRMT), and the outer mitochondrial membrane protein VDAC (α-VDAC/porin) as a mitochondrial loading control.
Mentions: To fully understand any transcription system, a critical parameter to know is the relative amounts of the basal transcription machinery in vivo. Our approach was to use immunoblotting to establish the levels of the mitochondrial transcription machinery on a total cell and mitochondrial basis. This method requires antibodies that are specific for their target, especially for homologous proteins that are very similar in size. Since h-mtTFB1 and h-mtTFB2 share a large degree of sequence similarity and are predicted to migrate similarly on polyacrylamide gels, it was unclear if antibodies we had generated would cross react with the paralogous protein and confound our analysis. Therefore, we first generated peptide antibodies against h-mtTFB1 and h-mtTFB2 (see Materials and Methods section) and determined their specificity by assessing reactivity toward purified full-length recombinant h-mtTFB1 and h-mtTFB2 by immunoblotting. We found that these antibodies were indeed highly specific for their targets showing no reactivity with the paralogous protein (Figure 1A). Having antibodies capable of specifically detecting each of the four human mitochondrial transcription proteins, as well as known amounts of corresponding recombinant proteins, allowed us to determine for the first time their relative abundance. We purified mitochondria from logarithmically growing HeLa cells and performed quantitative western blot analysis of POLRMT, h-mtTFA, h-mtTFB1 and h-mtTFB2 on known amounts of total mitochondrial protein. For each protein analyzed, multiple dilutions of total mitochondrial protein or total whole-cell lysate were probed in parallel with a dilution series of known amounts of recombinant protein to allow the amount of each transcription component relative to the total amount of mitochondrial lysate to be determined. Representative western blots and standard curves showing linearity of the assays employed are shown in Supplementary Figure S1.Figure 1.

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