The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3′ end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences. mitochondrion | RNA polymerase H uman mitochondria contain their own genetic material called mitochondrial DNA (mtDNA), a double-stranded circular molecule encoding 13 subunits of the respiratory chain, 22 tRNAs, and 2 rRNAs. The two strands are referred to as the heavy and light strand based on their buoyant density in cesium chloride gradients (1). Transcription from the light strand promoter (LSP) and heavy strand promoter is polycistronic and produces near genome-length transcripts, which are processed to release the individual RNA molecules (2, 3). MtDNA contains few noncoding sequences, with the exception of the control region, which harbors DNA elements required for initiation of transcription and DNA replication (Fig. 1A).Mitochondrial DNA replication initiates at two major sites, the origins of light and heavy (OriH) strand replication. The mitochondrial RNA polymerase generates the RNA primers required for initiation of DNA synthesis at both these sites (4-7). Primers required for OriH activation are produced by transcription initiated at LSP, and there must thus exist a mechanism that enables the transcription machinery to switch from genomelength transcription to primer formation. One model suggests that the primers are formed by cleavage of the primary LSP transcript by the mitochondrial RNA processing endonuclease (8, 9), but this idea has been questioned, because at least human mitochondrial RNA processing endonuclease is primarily localized to the nucleolus, where it is required for rRNA processing (10). Some years ago, we suggested an alternative model for primer formation at OriH. Using a reconstituted in vitro transcription system containing the mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factors A (TFAM) and B2 (TFB2M), we observed that a large fraction (up to 65%) of the transcription events initiated at LSP were prematurely terminated at a conserved DNA element within the control region, denoted conserved sequence block II (CSB II). The 3′ ends of these terminated transcription products coincided with the RNA-to-DNA transition sites mapped in vivo (5). The mechanism behind the observed transcription termination was not elucidated, but mutational analysis demonstrated ...
Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC O -tC nitro , we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.T he mtDNA is a double-stranded circular molecule that encodes 22 tRNAs, 2 rRNAs, and 13 subunits of the respiratory chain. Transcription is initiated from two sites, the light-and heavy-strand promoters (LSP and HSP1, respectively), and proceeds to produce near genome-length polycistronic transcripts, which are subsequently processed to generate the individual RNA molecules (1). Transcription from LSP also produces the RNA primers required for initiation of mtDNA replication at the origin of the heavy strand (2-4). In vivo experiments have identified a second transcription initiation site (HSP2) downstream of HSP1, but the sequence requirements of this promoter remain to be defined (5, 6).In budding yeast, the basic machinery for mtDNA transcription consists only of two factors: the mitochondrial RNA polymerase (Rpo41) and its accessory factor Mtf1, also denoted sc-mtTFB (1). In mammalian cells, it has been reported that mitochondrial transcription also requires the transcription factor A (TFAM), a high-mobility group-box (HMG) protein (7,8). TFAM plays a role as an mtDNA packaging factor that can bind, wrap, and bend DNA in a non-sequence-specific manner (9-12). TFAM also binds sequence-specifically to sites upstream (−15 to −35) of the HSP1 and LSP transcription start sites (13,14). The human mitochondrial RNA polymerase (POLRMT) is distantly related to the bacteriophage T7 RNA polymerase and has the capacity to recognize promoter elements (14). In combination, POLRMT, TFAM, and the mammalian Mtf1 homolog TFB2M can initiate transcription from a promoter-containing DNA fragment in vitro (15). TFB2M forms a heterodimeric complex with POLRMT and in...
In human mitochondria the transcription machinery generates the RNA primers needed for initiation of DNA replication. A critical feature of the leading-strand origin of mitochondrial DNA replication is a CG-rich element denoted conserved sequence block II (CSB II). During transcription of CSB II, a G-quadruplex structure forms in the nascent RNA, which stimulates transcription termination and primer formation. Previous studies have shown that the newly synthesized primers form a stable and persistent RNA–DNA hybrid, a R-loop, near the leading-strand origin of DNA replication. We here demonstrate that the unusual behavior of the RNA primer is explained by the formation of a stable G-quadruplex structure, involving the CSB II region in both the nascent RNA and the non-template DNA strand. Based on our data, we suggest that G-quadruplex formation between nascent RNA and the non-template DNA strand may be a regulated event, which decides the fate of RNA primers and ultimately the rate of initiation of DNA synthesis in human mitochondria.
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