The Conformational Dynamics of Yeast and Human Mitochondrial Transcription Initiation Revealed by Single-Molecule FRET Measurements

Abstract

The accurate and efficient transmission of genetic information is fundamental to cellular survival and normal physiological function, while defects in these processes can lead to severe diseases such as cancer. This study employs single-molecule Förster resonance energy transfer (smFRET) techniques to uncover the molecular principles and regulatory pathways governing critical steps in mitochondrial transcription and DNA replication. By revealing the intricate structural transitions and spatiotempo- ral control mechanisms underlying initiation-elongation transitions in mitochondrial transcription and clamp loading/unloading processes in DNA replication, the findings have broad implications. They not only deepen our understanding of the fidelity and error-correction mechanisms essential for minimizing mutations during gene expression and genome maintenance but also provide insights that could inform the prevention and treatment of diseases linked to genomic instability. Ultimately, this research lays the groundwork for developing novel strategies to enhance transcriptional and replicative accuracy at the molecular level, potentially leading to innovative biological and medical applications. In yeast mitochondrial transcription, the RNA polymerase Rpo41 and its initiation factor Mtf1 coor- dinate a series of conformational states that move from open promoter complexes to progressively more scrunched DNA arrangements as nascent RNA grows from +2 to +7 nucleotides. Rather than a smooth progression, this early phase is punctuated by backtracking, abortive initiation, and partial RNA release events, all observable in real time via smFRET. At a critical transition around +8, the tension built up in scrunched states is relieved, and the complex shifts abruptly into a stable elongation mode. Crucially, the flexible Mtf1 C-terminal tail is essential for stabilizing these scrunched intermediates, ensuring that only properly initiated transcripts proceed into productive elongation. This highlights how mitochon- drial transcription is finely tuned to ensure fidelity and efficiency before committing to full-length RNA synthesis. Human mitochondrial transcription with initiation factors (POLRMT, TFAM, TFB2M), shows a similarly intricate conformational ballet. TFAM bends the LSP promoter, enabling POLRMT to engage and open the duplex. As the polymerase adds nucleotides one by one, smFRET reveals that key struc- tural changes around the translocation steps rather than nucleotide incorporation itself. After the first few nucleotides, multiple elongation states emerge, and introducing a non-cognate NTP halts normal transi- tions. This demonstrates POLRMT’s innate proofreading capacity, aborting faulty transcripts early on. Thus, both yeast and human mitochondrial systems underscore the presence of kinetic and structural checkpoints that safeguard transcription accuracy. The studies also extend to DNA replication, focusing on how clamps like PCNA are loaded and unloaded from DNA. PCNA, a sliding clamp that enhances DNA polymerase processivity, must be precisely placed and removed to maintain genomic stability. Using smFRET, we identified key interme- diates during PCNA loading by RFC, confirming a multi-step model involving partial engagement and eventual ring closure around DNA. More importantly, we discovered that ATAD5-RLC is a specialized and potent PCNA unloader, surpassing other clamp-loader complexes. While ATP hydrolysis is often crucial for such mechanisms, ATAD5-RLC can initiate PCNA release with just ATP binding, although hydrolysis further enhances efficiency. This capacity to unload PCNA under varying conditions ensures that replication does not persist unnecessarily and that PCNA does not linger on DNA, potentially re- cruiting inappropriate factors and causing genomic instability. Collectively, these integrated findings highlight the power of single-molecule techniques in uncov- ering transient intermediates and subtle conformational shifts that govern complex molecular machines. By revealing that transcription initiation and elongation, as well as clamp loading and unloading, in- volve multiple branching pathways and regulatory checkpoints, this work deepens our understanding of fundamental molecular biology processes. The insights have broad implications: understanding the fidelity and regulatory mechanisms in transcription and replication can inform studies of mitochondrial dysfunction, human diseases such as cancer, and potential therapeutic strategies targeting transcription or replication machinery.