Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 25;45(2):861-874.
doi: 10.1093/nar/gkw1157. Epub 2016 Nov 29.

Human Mitochondrial Transcription Factors TFAM and TFB2M Work Synergistically in Promoter Melting During Transcription Initiation

Affiliations
Free PMC article

Human Mitochondrial Transcription Factors TFAM and TFB2M Work Synergistically in Promoter Melting During Transcription Initiation

Aparna Ramachandran et al. Nucleic Acids Res. .
Free PMC article

Abstract

Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has 'post-recruitment' roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from -4 to +1 in the open complex with all three proteins and from -4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar Kd values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery.

Figures

Figure 1.
Figure 1.
Transcription initiation activity of POLRMT, TFB2M and TFAM on the LSP DNA. (A) Sequence of the LSP DNA (−42 to +21) is shown. (B) The 24% polyacrylamide–4M urea gel image shows the abortives (2 to 6-mer) and the run off products (18 and 19-mer) after 10 min of transcription reaction at 25°C on the LSP DNA (1 μM) with individual and various combinations of POLRMT, TFB2M and TFAM (1 μM) and NTPs (ATP, GTP and UTP, 3′dCTP at 250 μM each) + [γ32P]ATP. (C) Salt concentration dependence of LSP DNA transcription by POLRMT+TFB2M in the presence and absence of TFAM. The transcription reactions were carried out as described in (B) under the indicated sodium glutamate salt concentrations. The abortives and run off products are shown. (D) The quantitation of total run-off (y-axis shown in log scale) and 2–6 mer abortives as a function of salt concentration. The experiment was performed once. (E) Transcription reactions were carried out as in (B) but with different concentrations of ATP and [γ32P]ATP for 15 minutes in presence and absence of TFAM. The 24% polyacrylamide–4M urea gel image shows the 2 mer, 3 mer and the slippage products. The catalytic efficiency (kcat/Km) was calculated by plotting the rate of RNA synthesis/enzyme concentration against the ATP concentration and fitting the curve to Hill Equation (Equation 2 in Materials and Methods). The experiment was performed twice. The average kcat/Km with the standard deviation in the presence of all three proteins is 64 ± 0.8 M−1 s−1 while in the absence of TFAM is 30 ± 13 M−1 s−1.
Figure 2.
Figure 2.
Interactions of the LSP DNA with TFAM, POLRMT and TFB2M measured by fluorescence anisotropy titrations. (A) The 5′-end fluorescein labeled LSP DNA (−41 to +10) was used in the fluorescence anisotropy titrations. (B) Binding of TFAM+POLRMT+TFB2M to the LSP DNA. Fluorescein-labeled LSP DNA (1 nM) was titrated with increasing concentration of equimolar mixture of TFAM+POLRMT+TFB2M and fluorescence anisotropy was recorded after excitation at 490 nm and emission at 514 nm. The plots show the fluorescence anisotropy change corrected for free DNA (anisotropy of 0.16). The binding data were fit to Equation (3). (C) Binding of the individual proteins to the LSP DNA. (D) Comparison of LSP DNA binding to TFAM alone and TFAM+POLRMT. (E) Comparison of LSP DNA binding to TFAM alone and TFAM+TFB2M. (F) Comparison of LSP DNA binding to POLRMT and POLRMT+TFB2M+ATP. Fluorescein labeled LSP DNA (1 nM) and 1 mM ATP was titrated with increasing concentration of an equimolar mixture of POLRMT+TFB2M. (G) The table summarizes the apparent Kd values and n (Hill coefficient) values from fits to the Hill equation. The Kd values shown are from two sets of independent experiments, n values are shown as an average. Associated error is the standard error of fitting.
Figure 3.
Figure 3.
FRET studies to investigate protein induced bending of the LSP DNA. (A) Representative fluorescence emission spectra of 10 nM LSP DNA (−41 to +10) with D–A pair in the upstream region show the dose dependent decreasing donor fluorescence intensity and increasing acceptor fluorescence intensity with addition of TFAM+POLRMT+TFB2M. The FRET efficiency was calculated using sensitized acceptor fluorescence calculated by the (ratio)A method (Materials and Methods and Supplementary Methods). (B) Comparison of upstream and downstream LSP DNA bending by TFAM. The LSP DNA with D–A pair in the upstream region and downstream region was titrated with TFAM. The y-axis shows the FRET efficiency. (C) Comparison of upstream LSP DNA bending by TFAM, TFAM+POLRMT and TFAM+POLRMT+TFB2M. (D) Comparison of downstream LSP DNA bending by POLRMT+TFB2M, POLRMT+TFAM and POLRMT+TFAM+TFB2M. The errors were calculated from two independent experiments.
Figure 4.
Figure 4.
2-AP fluorescence mapping to determine the melted DNA region in the LSP promoter and the roles of TFAM, TFB2M,and POLRMT in open complex formation. (A) Sequence of the LSP promoter fragment (−42 to +21) is shown with the positions of 2-AP substitutions in red and the numbering is relative to the transcription initiation site indicated as +1. (B) Structures of 2-AP:dT and 2-AP:dC base pairs (45,46). (C) The table indicates the individual base changes to 2-AP, the resulting base pairs, and the transcriptional activity of the singly 2-AP labeled LSP promoters. (D) The bar chart shows the fold increase in 2-AP fluorescence (excitation at 315 nm and emission at 370 nm) in the LSP promoters (100 nM) substituted with 2-AP relative to free 2-AP labeled LSP promoter in the presence of TFAM+POLRMT+TFB2M (equimolar 150 nM). The cartoon summarizes the FRET and 2-AP results showing the open complex generated with all three proteins (TFAM, light blue; TFB2M, dark blue; POLRMT, orange). (E) ATP (0.5 mM) was added after addition of equimolar TFAM+POLRMT+TFB2M to each of the 2-AP labeled LSP promoter DNAs. The cartoon summarizes the FRET and 2-AP results showing the initiation complex with all three proteins and ATP. (F) The bar chart shows the fold increase in 2-AP fluorescence at the -1NT position with POLRMT+TFAM and POLRMT+TFB2M with and without ATP in comparison to all three proteins and ATP. The numbers over the bars are the fold increase in fluorescence intensity over the free DNA fluorescence. The cartoon shows the two possible semi-open complexes of POLRMT+TFAM and POLRMT+TFB2M on LSP. The error bars in the above experiments are from two independent experiments.
Figure 5.
Figure 5.
A minimal model of transcription initiation on LSP in human mitochondria. The cartoon shows that under high TFAM conditions, the LSP DNA region is bound to two TFAM molecules, one on the upstream and the other on the downstream LSP region. Similarly, under low TFAM conditions, one TFAM molecule is bound to the upstream site. As shown by our FRET studies and those in the literature (16), the TFAM bound upstream DNA is more severely bent than the TFAM bound downstream region. Transcription initiates when the POLRMT+TFB2M displaces the TFAM molecule bound to the downstream initiation region or it directly binds to the protein-free initiation region. The resulting POLRMT+TFAM+TFB2M promoter DNA complex contains a U-turn upstream DNA bend and an ∼80o downstream DNA bend that brings all three proteins in close vicinity to one another to form a compact open complex in which the DNA is melted from −4 to +1. The binding of the initiating ATP converts the open complex to the initiation complex in which the DNA bubble is expanded from −4 to +3.

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

References

    1. Shadel G.S. Expression and maintenance of mitochondrial DNA: new insights into human disease pathology. Am. J. Pathol. 2008;172:1445–1456. - PMC - PubMed
    1. Shutt T.E., Shadel G.S. A compendium of human mitochondrial gene expression machinery with links to disease. Environ. Mol. Mutagen. 2010;51:360–379. - PMC - PubMed
    1. Cermakian N., Ikeda T.M., Miramontes P., Lang B.F., Gray M.W., Cedergren R. On the evolution of the single-subunit RNA polymerases. J. Mol. Evol. 1997;45:671–681. - PubMed
    1. Davanloo P., Rosenberg A.H., Dunn J.J., Studier F.W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 1984;81:2035–2039. - PMC - PubMed
    1. Deshpande A.P., Patel S.S. Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase. Biochim. Biophys. Acta. 2012;1819:930–938. - PMC - PubMed

Publication types

Feedback