Biology subjects

Heiss, F. B.

Publications and source records attributed to Heiss, F. B..

2 recordsLinked to original sources

bioRxiv2021

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This dock II domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

biochemistry↗

bioRxiv2019

DNA origami-based single-molecule force spectroscopy unravels the molecular basis of RNA Polymerase III pre-initiation complex stability

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor compound the fundamental core of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional intiation factor Bdp1, which is unique to the RNA polymerase (RNAP) III sytem, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain, that arises from DNA compaction and transcriptional activity, on the efficiency of initiation complex formation. We made use of a new nanotechnological tool - a DNA origami-based force clamp - to follow the assembly of human initiation complexes in the Pol II and Pol III system at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is necessary and sufficient to stabilise TBP on a strained RNAP II promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of Pol III genes for the first time.

biophysics↗