Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA

Abstract

Characterization of the basic transcription machinery of mammalian mitochondrial DNA (mtDNA) is of fundamental biological interest and may also lead to therapeutic interventions for human diseases associated with mitochondrial dysfunction. Here we report that mitochondrial transcription factors B1 (TFB1M) and B2 (TFB2M) are necessary for basal transcription of mammalian mitochondrial DNA (mtDNA). Human TFB1M and TFB2M are expressed ubiquitously and can each support promoter-specific mtDNA transcription in a pure recombinant in vitro system containing mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A. Both TFB1M and TFB2M interact directly with POLRMT, but TFB2M is at least one order of magnitude more active in promoting transcription than TFB1M. Both factors are highly homologous to bacterial rRNA dimethyltransferases, which suggests that an RNA-modifying enzyme has been recruited during evolution to function as a mitochondrial transcription factor. The presence of two proteins that interact with mammalian POLRMT may allow flexible regulation of mtDNA gene expression in response to the complex physiological demands of mammalian metabolism.

3D model of RNA polymerase and bidirectional transcription

“…LSP was mapped between 16183bp and inside region of the tRNA pro [16,33,35 ]. These results of the in-vitro generated transcription products presented in this study are also in good agreement with the previous reports [13,17,20,30] of the in-vivo transcription initiation products from the HSP/LSP containing the mt D-loop region spanning at one end of tRNA leu and the other end of tRNA thr . All of these results discussed here indicate that the LES invitro reconstituted transcription complex was able to transcribe bidirectionally from both Hstrand-promoter (HSP) and light-strand-promoter (LSP) which lie within the D-loop segment.…”

“…Mitochondrial D-loop region; In-vitro transcription; mt RNA polymerase; gel-mobility shift assay; 3D model Both human and mouse mitochondrial (mt) in-vitro reconstituted transcription system and mt lysate were shown to transcribe bidirectionally from D-loop region containing H-strandpromoter/L-strand-promoter (HSP/LSP) of mitochondrial genome .These transcripts ranged between >100bp and >3000bp in both human and mouse [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The D-loop distal genes were also transcribed in this system [16].…”

“…The ideal human transcription complex [13] was described to constitute RNA polymerase, transcription factor TFB1M (or TFB2M having weaker activity than TFB2M), LSP/HSP template (1-741bp) and mt general transcription factor TFAM. The mouse homologs (or orthologs) mTfb1m and mTfb2m were identified [13]. The transcription termination was reported to occur by 34kd mt-TERM/mtTERF DNA binding protein [14,15], although 24kd Tf1 transcription factor, 48kd TAS-A protein (which binds to termination associated sequence of mt D-loop region) as well as other putative binding proteins (TAS-B, -C, -D, -E, -F, -G; A/B and F/G which bind to partially overlapping sites) in the mt D-loop segment were reported [15,16].…”

“…. In the in-vitro reconstituted transcription system (isolated by the heparin-sepharose chromatography), the HSP/LSP region (where both major and minor promoters lie within close proximity) of the mitochondrial Dloop segment in both human and mouse were reported previously to initiate transcription products which ranged between >100bp to 700bp runoff transcripts [13][14][15][16][17]19,20,29,30], although the mitochondrial lysate transcribed >2050bp ->3000bp [16] in an in vitro assay system. The mitochondrial lysate produced comparatively larger transcripts (>4-5kb) from HSP/LSP fused with larger human mitochondrial DNA segment [16].…”

“…RNase MRP cleaves mRNA substrate at an adjacent conserved decamer sequence 5′-CGACCCCUCC-3′ (relative to CSB2 in D-loop), whereas RNase P is involved in 5′ and 3′ end-processing of mt tRNA [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. This conserved decamer sequence 5′-CGACCCCUCC-3′ is also present in MRP RNA [3,4,7,[11][12][13]. …”