The mitochondrial transcription machinery is a rather simple system, consisting of mitochondrial RNA polymerase, the core protein, mitochondrial transcription factor A
(TFAM) which acts as an activator, and TFB1M or TFB2M which are needed for initiation. TFB2M has been shown to be primarily the transcription factor (Cotney et al. 2007). In 1985 it was reported that a transcription factor (or factors) is needed for specific initiation of transcription at HSP and LSP (Fisher and Clayton 1985) and the same year Hixson and Clayton (1985) established that specific residues at the transcription initiation sites are needed for transcription initiation from either HSP or LSP. Subsequently Fisher
et al. (1987) established that binding of a transcription factor to a regulatory element,
independent of orientation, is required for successful promoter selection. The relevant factor, human TFAM protein of 24.4 kDa, was finally purified and characterized in 1988 (Fisher and Clayton 1988). Recently, Shutt et al. (2010) reported that specific transcription initiation can take place in vitro independent of TFAM from both LSP and HSP1.
2.2.1.1 Mitochondrial RNA polymerase
Mitochondrial RNA polymerase activity was first characterized by Shuey and Attardi (1985). Masters et al. (1987) first established the homology between the yeast mitochondrial RNA polymerase and those of bacteriophages T3 and T7 whereas no homology was detected between the yeast mitochondrial enzyme and E. coli RNA polymerase. Tiranti et al. (1997) identified the nuclear gene on chromosome 19p13.3. coding for the human mitochondrial RNA polymerase (h-mtRPOL, here called POLRMT) which is a protein of 1230 amino acids. Prieto-Martin et al. (2001) suggested that additional factors are needed for transcription initiation, since POLRMT, either alone or together with TFAM or the termination factor MTERF (see below, section 2.2.2), was not able to initiate transcription in vitro. Note also the recent finding of Shutt et al. (2010), showing that TFAM is not necessary to initiate transcription in vitro from LSP or HSP1.
2.2.1.2 Mitochondrial transcription factor A
TFAM belongs to the high mobility group (HMG)–box family of DNA-binding proteins (Parisi and Clayton 1991), and is able to alter mtDNA structure, condensing, unwinding and bending it (Fisher et al. 1992) which in turn might facilitate transcription initiation. TFAM protein has two HMG-box domains with a 27 amino acid (aa) linker region between them and a 25 aa C-terminal tail that has been established to be important for accurate DNA recognition, and is limiting for transcriptional activation (Dairaghi et al. 1995). Knocking out murine Tfam leads to a decrease in mtDNA copynumber in heterozygous mice and in homozygous mice the knockout is embryonic lethal with massive depletion of mitochondrial DNA (Larsson et al. 1998). These findings clearly show that TFAM has an important role in mtDNA maintenance and is also an essential protein for embryonic development (Larsson et al. 1998).
TFAM is important in the initiation of mitochondrial transcription, since human mitochondrial RNA polymerase needs TFAM to recognize the promoters of human mitochondrial DNA. TFAM is a rather typical HMG protein in many respects e.g. it prefers binding oxidatively damaged mitochondrial DNA (Yoshida et al. 2002), is able to recognize cisplatin damaged DNA where it induces bends (Chow et al. 1994, Chow et al. 1995). TFAM binds mtDNA showing no sequence specificity (Fisher et al. 1989, Fisher
et al. 1992). The TFAM monomer also binds four-way DNA junctions for which it needs
both of the HMG-box domains (Ohno et al. 2000). TFAM, like many other HMG-box proteins, can be acetylated: Dinardo et al. (2003) reported that TFAM is acetylated at one lysine residue. Ohgaki et al. (2007) reported that the C-terminal tail of TFAM strengthens its binding to mtDNA. The evidence presented by Shutt et al. (2010) that transcription can be initiated in vitro from LSP and HSP1 independently of TFAM, raises questions concerning the primary role of TFAM in mitochondrial transcription.
2.2.1.3 Mitochondrial transcription factor B
A human counterpart of Saccharomyces cerevisiae mitochondrial transcription factor B was first reported by McCulloch et al. (2002). Human “mtTFB” was shown to bind mtDNA in a non-sequence-specific manner. McCulloch et al. (2002) showed that, in
vitro, mtTFB (now designated TFB1M) and TFAM together are able to activate
transcription from the human mitochondrial light-strand promoter. TFB1M can bind S- adenosylmethionine and shows homology to N6 adenine RNA methyltransferases methylating the N6 position of adenine in specific nucleotides in rRNA (McCulloch et al. 2002). This was the first report of a transcription factor related to an RNA-modifying enzyme (McCulloch et al. 2002).
Falkenberg et al. (2002) named two novel ubiquitously expressed transcription factors needed to initiate mammalian mitochondrial transcription as TFB1M and TFB2M: TFB1M is identical to the mtTFB identified by McCullogh et al. (2002). Falkenberg et
al. (2002) used purified recombinant versions of the mitochondrial proteins and
suggested that the minimum requirement for transcription from both heavy and light strand human mtDNA promoters consists of a protein complex of TFB1M or TFB2M, TFAM and the mitochondrial RNA polymerase. TFB2M is more active in transcription activation than TFB1M but is also related to bacterial rRNA methyltransferase (Falkenberg et al. 2002). Seidel-Rogol et al. (2003) reported that TFB1M has two functions: a role in transcription and also as an rRNA methyltransferase, which can methylate a conserved stem-loop both in bacterial 16S rRNA and in the homologous human 12S rRNA molecule. Cotney et al. (2007) established, using cultured cells, that TFB2M is primarily the transcription factor, as over-expression of TFB2M induces an approximately 2-fold increase in overall mitochondrial transcript levels whereas TFB1M has no such effect. Using cultured cells over-expressing TFB1M Cotney et al. (2007) also first presented in vivo evidence that TFB1M is the primary human mitochondrial 12S rRNA methyltransferase. Furthermore, Cotney et al. (2009) showed that TFB1M and TFB2M collaborate in mitochondrial biogenesis.
Tfb1m and Tfb2m, the murine TFB1M and TFB2M homologues, are ubiquitously expressed (Rantanen et al. 2003). Most metazoans seem to have two TFBM genes (Rantanen et al. 2003, Cotney and Shadel 2006). Cotney and Shadel (2006) reported that the two TFBM genes found in metazoans arise from a gene duplication event that took place before the divergence of fungi and metazoans in evolution, and in some organisms the selective pressure finally led to loss one of the genes.
Human TFB1M and TFB2M are both capable of binding the C-terminal tail of TFAM, the region that is needed for the activation of transcription (McCulloch et al. 2003). Human TFB1M co-immunoprecipitates with human POLRMT (McCulloch et al. 2003) indicating that it forms a link between the human TFAM and POLRMT which would further explain the initiation of transcription in human mtDNA (McCulloch et al. 2003). As TFB1M co-immunoprecipitates with POLRMT and in vitro has been shown to activate transcription, it is still possible that TFB1M has a role also in transcription which remains to be elucidated.
TFAM is essential in transcription initiation and it is required for POLRMT /TFB2M to be able to recognize the promoter (Gaspari et al. 2004). Gaspari et al. (2004) proposed that TFAM binds mtDNA inducing a structural change, enabling the POLRMT /TFB2M complex to recognize the promoter sequence. Sologub et al. (2009) showed that TFB2M facilitates promoter melting but is not a limiting factor for protein recognition. They also proposed that TFB2M has a role as a transient component of the catalytic site of the transcription initiation complex since it interacts with the priming substrate (Sologub et
al. 2009). Lodeiro et al. (2010) showed using transcription factors A and B2, which were
isolated from Escherichia coli, that both of them are needed for open complex formation which is the rate-limiting step for production of the first phosphodiester bond whereas the subsequent steps require only TFB2M. Litonin et al. (2010) established that only TFAM and TFB2M are needed for successful transcription in vitro whereas, as mentioned above, Shutt et al. (2010) have presented data indicating that in vitro transcription initiation can occur independently of TFAM.