Structural basis of human mitochondrial transcription initiation and processive elongation

In eukaryotic cells, mitochondria produce the vast majority of ATP, the universal energy currency of life. To do so, they maintain a highly reduced genome as well as the molecular machinery necessary for its expression. Transcription in mitochondria is carried out by a dedicated mitochondrial RNA polymerase (mtRNAP), which is related to single-subunit RNA polymerases (RNAPs) found in bacteriophages. In contrast to these self-sufficient enzymes, however, mtRNAP requires additional protein factors for all steps of transcription, suggesting a complex regulation. Moreover, it also produces the RNA primers necessary to initiate DNA synthesis, placing this enzyme at the heart of mitochondrial gene expression and organelle maintenance. Structures of mtRNAP have provided a first glimpse at the central actor orchestrating these important processes, but the mechanistic principles governing the individual steps of mitochondrial transcription remain poorly understood. In this study, we expand our understanding of these processes by investigating the structural basis of transcription initiation and processive elongation, two steps of regulatory importance. To initiate transcription, mtRNAP associates with the two initiation factors TFAM and TFB2M and promoter DNA to form an initiation complex (IC). Here, I present the structure of human TFB2M at 1.75 A resolution and of the human initiation complex at 4.5 A resolution. Together with published structures of mtRNAP and TFAM, this allows for construction of a pseudo- atomic model of the IC. The structures reveal how mtRNAP is recruited to the promoter by TFAM and suggest that TFB2M induces a rearrangement in mtRNAP to facilitate promoter opening. The open complex is further stabilized by interactions between TFB2M and the melted non-template DNA strand. Structural comparisons demonstrate that transition to elongation is accompanied by a profound re-arrangement of the upstream DNA. Following initiation, mtRNAP associates with the elongation factor TEFM for processive transcription elongation. This factor enables mtRNAP to transcribe through a G-quadruplex forming sequence in the mitochondrial genome, which otherwise leads to transcription termination and primer formation for replication. However, the mechanistic basis for this anti- termination activity of TEFM is unknown. Here, I present crystal structures of the human TEFM domains and, in a collaborative effort with the Temiakov Lab, we functionally define their roles in transcription. In addition, I have determined the structure of an anti-termination complex, comprised of the functional domains of TEFM bound to transcribing mtRNAP. These structures demonstrate that TEFM stabilizes the elongation complex by enclosing the downstream DNA in a “sliding clamp” and by interacting with the non-template strand in the transcription bubble. Moreover, these data suggest that TEFM prevents formation of the G-quadruplex in the RNA exit path, thereby mediating the switch between transcription and DNA replication. Taken together, these results greatly advance our understanding of mitochondrial transcription and elucidate the mechanistic basis for the factor dependence of mtRNAP. Furthermore, they provide a framework for future studies aimed at deciphering the regulatory mechanisms of transcription and DNA replication in human mitochondria.