Kugel, J. F. | bioRxiv Science

bioRxiv2025

Single-molecule microscopy reveals that TFIIE subunits dynamically interact with preinitiation complexes in a manner controlled by TFIIH

Transcription by RNA polymerase II (Pol II) requires general transcription factors that bind with Pol II at the promoters of protein-coding genes to form preinitiation complexes (PICs). Among these is TFIIE, which recruits TFIIH to the PIC and stimulates the kinase and translocase activities of TFIIH, thereby regulating the fate of formed PICs. In this study, we used a purified reconstituted human Pol II transcription system and single molecule total internal reflection fluorescence (smTIRF) microscopy to monitor TFIIE binding dynamics in PICs under different conditions in real time. We observed highly dynamic interactions of the two subunits of TFIIE (TFIIE and TFIIE{beta}) with PICs. Measurement of rate constants for on/off binding of each subunit suggest they behave asynchronously. TFIIH exclusion increased the rates of association and dissociation for both subunits, with the strongest effect on TFIIE. Despite stabilization of TFIIE by TFIIH the TFIIE subunits remain dynamic in PICs. Additionally, two disease-related TFIIE{beta} point mutations destabilized TFIIE{beta} and altered its kinetic behaviors within PICs. Our results contribute to an emerging model that PICs are not static assemblies and highlight important connections between the structural arrangement and kinetic behaviors of GTFs in PICs.

biochemistry↗

bioRxiv2020

miR-206 knockout shows it is critical for myogenesis and directly regulates newly identified target mRNAs

The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six putative targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.

molecular biology↗