Biology subjects

Muller, C. W.

Publications and source records attributed to Muller, C. W..

6 recordsLinked to original sources

bioRxiv2025

High-resolution mapping of human RNA polymerase III reveals transcription termination as a rate-limiting step

Transfer RNA (tRNA) molecules play a central role in the flow of genetic information, translating nucleic acid sequences into the functional protein portfolio of an organism. Despite the high abundance of tRNAs in cells, studying their biology remains challenging due to the repetitive nature of genomic sequences and the numerous modifications of mature molecules. tRNA genes (tDNAs) are transcribed exclusively by RNA polymerase III (RNAPIII), which has been recently linked to rare genetic disorders but also longevity. Here, we present the first mapping of actively transcribing RNAPIII in the human, using K562 cell line and UV-crosslinking followed by stringent purification. Our data reveals high variability in expression across tDNAs, common occurrences of transcriptional read-through, and unique transcription dynamics across transcription units, with the lowest kinetics associated with transcription termination. Further analysis revealed that release of the nascent transcript is a critical step in tRNA transcription. Unexpectedly, shorter terminators promote more efficient termination, which becomes the rate-limiting step for human RNAPIII transcription of highly expressed tDNAs. Our dataset provides insights into actively transcribing RNAPIII dynamics in human cells, allowing for a comprehensive analysis of genomic loci transcribed by RNAPIII. Our high resolution data and resulting kinetic information reveal that human RNAPIII contradicts the paradigm that initial steps of transcription are the main determinants of its output. This is significant as RNAPIII is commonly used to transcribe synthetic RNA constructs, such as short hairpin RNAs or guide RNAs used in gene editing technologies. With our study we offer a functional framework for the analysis of RNAPIII activity, addressing the need for comprehensive understanding of RNAPIII transcription fueled by a growing number of described genetic disorders caused by mutations in this enzyme.

molecular biology↗

bioRxiv2024

Structural insights into distinct mechanisms of RNA polymerase II and III recruitment to snRNA promoters

RNA polymerase III (Pol III) is specialized in the transcription of short, essential RNAs, including the U6 small nuclear RNAs (snRNAs). At U6 snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc) forming, together with a Brf2-containing TFIIIB complex, a transcriptionally competent pre-initiation complex (PIC). Additionally, SNAPc is responsible for the recruitment of Pol II at the remaining snRNAs genes (U1, 2, 4 and 5), representing a unique example of a multi subunit transcription factor shared among different RNA Polymerases. The mechanism of SNAPc cross-polymerase engagement and the role of the SNAPC2 and SNAPC5 subunits in transcription remain poorly defined. Here, we present cryo-EM structures of the full-length SNAPc-containing Pol III PIC assembled on the U6 snRNA promoter in the open and melting states at 3.2-4.2[A] resolution. Comparative structural analysis revealed unexpected differences with the yeast PIC and revealed the molecular basis of selective and structurally distinct SNAPc engagement within Pol III and Pol II PICs. Harnessing crosslinking mass spectrometry, we also localize the SNAPC2 and SNAPC5 subunits in proximity to the bound promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.

biochemistry↗

bioRxiv2024

Structural and kinetic insights into tRNA promoter engagement by yeast general transcription factor TFIIIC

Transcription of tRNA genes by RNA polymerase III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryo-EM and single-molecule FRET (smFRET) to reveal structural and real-time kinetic insights into how the 520 kDa yeast TFIIIC complex engages A- and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of {tau}A and {tau}B subcomplexes bound to the A- and B-box were obtained at 3.7 and 2.5 [A] resolution, respectively, while cryo-EM single particle mapping determined the specific distance and relative orientation of the {tau}A and {tau}B subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of {tau}A and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

biochemistry↗

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗

bioRxiv2021

An integrated model for termination of RNA polymerase III transcription

RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

genomics↗

bioRxiv2018

Structural rearrangement of TFIIS- and TFIIF/TFIIE-like subunits in RNA polymerase I transcription complexes

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-EM structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-electron microscopy structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 [A] resolution that provide additional insight into the functional interplay between the TFIIE/TFIIF-like A49-A34.5 heterodimer and the TFIIS-like subunit A12.2 present in Pol I. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our work suggests a regulatory mechanism of Pol I transcription where the association of the A49-A34.5 heterodimer to Pol I is regulated by subunit A12.2, thereby explaining in vitro biochemical and kinetic data.

molecular biology↗