Cryo-EM Reveals RECQ5's Regulatory Role in RNAPII-Mediated Transcription

K. Skubnik1, M. Sebesta1, J. Moravcova1, J. Novacek1, R. Stefl12

1CEITEC–Central European Institute of Technology, Masaryk University; Brno

2National Centre for Biomolecular Research, Faculty of Science, Masaryk University; Brno

karel.skubnik@ceitec.muni.cz

In eukaryotic nuclei, the synthesis of mRNA is carried out by RNA polymerase II (RNAPII), a crucial enzyme in transcriptional processes. RECQ5 helicase, a general elongation factor, associates with RNAPII and controls its movement along genes. Despite its significance, the precise mechanism through which RECQ5 regulates RNAPII movement remained unknown.

Here, we present the details of the interaction between RECQ5 and RNAPII determined by cryo-electron microscopy. Single-particle analysis with a resolution of 3 Å revealed near-atomic level details of the interaction between the RECQ5 helix and RNAPII DNA. Additionally, cryo-EM imaging showed that the RNAPII with RECQ5 complex formed large, spherical objects resembling condensates. Correlative light electron microscopy confirmed the presence of both fluorescently labeled RNAPII and RECQ5 within these objects. Subtomogram averaging revealed the organization of condensates, further enhancing our understanding of their functional assembly. The subtomogram averaging model of RNAPII with RECQ5 reached a resolution of 7 Å, confirming the observed interaction between the RECQ5 helix and RNAPII DNA across analytical methods.

Our study emphasizes RECQ5's crucial role in modulating transcriptional processes by using the brake-helix to regulate RNAPII movement along genes. Additionally, we discovered that condensates contain tens to hundreds of RNAPII molecules with RECQ5. The study provides valuable insights into how transcriptional machinery functions in eukaryotic nuclei.All papers should be prepared by using British English.