title: Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA.
abstract: Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Brown
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.
authors.forename: Ariana N
authors.initials: AN
authors.lastname: Tkachuk
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.
authors.forename: David A
authors.initials: DA
authors.lastname: Clayton
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D041701:Swiss 3T3 Cells
chemical_list: D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D000077278:RNA, Mitochondrial; C081930:Tfam protein, mouse; D012313:RNA; D012343:RNA, Transfer