The human mitochondrial transcription termination factor (mTERF) is fully active in vitro in the non-phosphorylated form.
The human mitochondrial transcription termination factor (mTERF) is a 39-kDa protein that terminates transcription at the 3'-end of the 16 S rRNA gene and thereby controls expression of the ribosomal transcription unit of mitochondrial DNA. The transcription termination activity of human mTERF has been notoriously difficult to study in vitro, and it has been suggested that the activity of the protein is regulated by posttranslational modifications or by protein polymerization. We here characterize the activity of recombinant human mTERF expressed in insect cells. We observed that mTERF efficiently promotes sequence-specific termination in a completely recombinant and highly purified in vitro system for mitochondrial transcription. The termination activity has a distinct polarity, and we observed complete transcription termination when the mTERF-binding site is oriented in a forward position relative the heavy strand promoter but only partial transcription termination when the binding site is in the reverse position. We analyzed the biochemical characteristics of the active mTERF protein and found that it is a stable monomer at physiological salt concentration. Structural analysis, including phosphostaining, two-dimensional electrophoresis, and electrospray mass spectrometry, detected no evidence of phosphorylation. We conclude that the monomeric human mTERF is fully active in its non-phosphorylated form and that the protein does not require additional cellular factors to terminate mitochondrial transcription in vitro.
Asin-Cayuela
Jordi
J
Department of Medical Nutrition, Karolinska Institutet, Novum, Huddinge Hospital, S-141 86 Huddinge, Sweden.
Schwend
Thomas
T
Farge
Géraldine
G
Gustafsson
Claes M
CM
D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors; D002478:Cells, Cultured; D006801:Humans; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008970:Molecular Weight; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D018411:Spodoptera; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D050976:Basic-Leucine Zipper Transcription Factors; C105178:MTERF1 protein, human; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors