Mechanism of Transcription Anti-termination in Human Mitochondria

Abstract

Summary In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA – the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the EC. Binding of TEFM to the DNA forms a downstream “sliding clamp”, providing high processivity to the elongation complex. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA.

TEFM regulates both transcription elongation and RNA processing in mitochondria

“…Using Flp-In T-REx 293 cells, we generated a stable cell line that constitutively expressed human TEFM as a fusion protein containing a carboxyterminal BirA*-HA tag (TEFM-BirA*). As expected, our results confirmed that TEFM interacts with POLRMT ( Fig 6), as previously reported [16,17,19]. In addition, we identified a number of RNA processing-related proteins that interacted with TEFM, including the RNase P complex (MRPP1-3), GRSF1, FASTKD5, ELAC2, mtPAP, and the components of mitochondrial RNA degradosome (SUPV3L1-PNPase) complex (Fig 6), which are all reported to be localized in or adjacent to RNA granules [29,30,37,40,41,57].…”

“…The newly transcribed RNA remains associated with the CSB region, where it forms an R-loop that is resistant to treatment by RNase A and RNase T1 [24][25][26]. Interestingly, TEFM has been reported to regulate the generation of replication primers in human mitochondria [27] by helping POLRMT to bypass the highly structured CSBII region [19,20] and abolish R-loop formation [17]. Results from an in vitro study have been interpreted to support a model where TEFM serves as a molecular switch that coordinates the balance between mtDNA transcription for replication primer formation and gene expression [27], whereas another in vitro study argues that TEFM is a general unspecific transcription elongator needed for mtDNA gene expression [17].…”

Mechanisms of mammalian mitochondrial transcription

“…66 Importantly, binding of TEFM is incompatible with TFB2M binding, and consistently TEFM is unable to associate with POLRMT during initiation. 66 Furthermore, the transition to elongation appears to involve substantial rearrangements in the topology of the DNA upstream of the POLRMT active site. 31 The increase in processivity resulting from association of TEFM and POLRMT can overcome transcriptional termination occurring at various sites in the mtDNA and facilitate bypass of oxidative lesions.…”

“…The TEFM crystal structure confirms that it dimerizes and reveals that each monomer contains two tandem helix–hairpin–helix domains that are dispensable to enhance POLRMT processivity as well as an RNase H‐like fold that mediates the activities relevant to transcriptional elongation. Moreover, dimerization is essential for its association with POLRMT . A crystal structure of a transcription elongation complex showing TEFM complexed with POLRMT shows that TEFM binds POLRMT near the RNA exit channel, forming a sliding clamp that presumably increases the processivity of POLRMT (Figure d) .…”

“…66 A crystal structure of a transcription elongation complex showing TEFM complexed with POLRMT shows that TEFM binds POLRMT near the RNA exit channel, forming a sliding clamp that presumably increases the processivity of POLRMT (Figure 2d). 66 Importantly, binding of TEFM is incompatible with TFB2M binding, and consistently TEFM is unable to associate with POLRMT during initiation. 66 Furthermore, the transition to elongation appears to involve substantial rearrangements in the topology of the DNA upstream of the POLRMT active site.…”