Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Sequence-specific DNA looping by mitochondrial transcription factor A (TFAM)

Mitochondrial transcription factor A (TFAM) is an abundant human mitochondrial High Mobility Group Box (HMGB) protein. Similar to several human nuclear HMGB proteins, TFAM has two HMGB domains that facilitate DNA binding and bending (Ngo et al., 2014; Rubio-Cosials et al, 2011; Ngo et al., 2011). TFAM is an architectural protein known to play an essential role in shaping and maintaining the mitochondrial DNA (mtDNA). It is also known to be involved in regulating mitochondrial transcription. Sequence-specific binding of TFAM to DNA upstream of the light-strand promoter (LSP) leads to bending that correlates with transactivation of this promoter. The role for TFAM and corresponding mechanism at other promoters is likely different than observed at LSP (Lodeiro etal., 2012). For example, transcription from HSP1 uses far more upstream sequence than required at LSP. Here we use a dual promoter construct containing both LSP, HSP1 and the natural inter-promoter region to understand the role of the IPR on transcription. We show that the IPR contributes to TFAM transactivation of HSP1. Removal of the carboxy-terminal tail of TFAM (TFAM-ΔCT26) leads to a complete loss of transactivation of HSP1 with only minimal effects on LSP. By using atomic force microscopy (AFM), we observe that TFAM preferentially binds to the IPR. In addition, we find that at low concentrations of TFAM, this binding leads to formation of DNA loops in a region consistent with the IPR. Interestingly, under the same conditions, TFAM-ΔCT26 fails to produce DNA loops. Importantly, at higher concentration both TFAM and TFAM-ΔCT26 are equally efficient at DNA compaction. Taken together, our results are consistent with sequence-specific DNA looping contributing to TFAM transactivation of HSP1, suggesting that unique mechanisms are employed for TFAM-dependent transcription at LSP and HSP1.

    Ngo, H.B., G.A. Lovely, R. Phillips, and D.C. Chan (2014). Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation. Nat Commun, 5: p. 3077.

    Rubio-Cosials, A., J.F. Sidow, N. Jimenez-Menendez, P. Fernandez-Millan, J. Montoya, H.T. Jacobs, M. Coll, P. Bernado, and M. Sola (2011). Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter. Nat Struct Mol Biol, 18: p. 1281-9.

    Ngo, H.B., J.T. Kaiser, and D.C. Chan (2011). The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA. Nat Struct Mol Biol, 18: p. 1290-6.

    Lodeiro, M.F., A. Uchida, M. Bestwick, I.M. Moustafa, J.J. Arnold, G.S. Shadel, and C.E. Cameron (2012.). Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro. Proc Natl Acad Sci U S A, 2012. 109: p. 6513-8.

Divakaran Murugesapillai1
Maria F. Lodeiro2
L. James Maher III3
Craig E. Cameron2
Mark C. Williams1

1 Northeastern University
Department of Physics,
Boston, MA 02115, USA
2 Pennsylvania State University
Department of Biochemistry and Molecular Biology
University Park
PA 16802, USA
3 Mayo Clinic College of Medicine
Department of Biochemistry and Molecular Biology
Rochester, MN 55905, USA