title: Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation.
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 Kdvalues, 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical school, Piscataway, NJ 08854, USA.
authors.forename: Aparna
authors.initials: A
authors.lastname: Ramachandran
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical school, Piscataway, NJ 08854, USA.|Graduate School of Biomedical Sciences, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Urmimala
authors.initials: U
authors.lastname: Basu
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical school, Piscataway, NJ 08854, USA.
authors.forename: Shemaila
authors.initials: S
authors.lastname: Sultana
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical school, Piscataway, NJ 08854, USA.
authors.forename: Divya
authors.initials: D
authors.lastname: Nandakumar
authors.affiliation: Department of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Medical school, Piscataway, NJ 08854, USA patelss@rutgers.edu.
authors.forename: Smita S
authors.initials: SS
authors.lastname: Patel
mesh_terms: D001665:Binding Sites; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*; D061785:Transcription Initiation, Genetic
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D046912:Multiprotein Complexes; C499631:TFAM protein, human; D014157:Transcription Factors; D008780:Methyltransferases; C462443:TFB2M protein, human