Biology subjects

Cramer, P.

Publications and source records attributed to Cramer, P..

5 recordsLinked to original sources

bioRxiv2018

Structure of transcribing RNA polymerase II-nucleosome complex

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through chromatin. Pol II passage is impaired by nucleosomes and requires elongation factors that help Pol II to efficiently overcome the nucleosomal barrier1-4. How the Pol II machinery transcribes through a nucleosome remains unclear because structural studies have been limited to Pol II elongation complexes formed on DNA templates lacking nucleosomes5. Here we report the cryo-electron microscopy (cryo-EM) structure of transcribing Pol II from the yeast Saccharomyces cerevisiae engaged with a downstream nucleosome core particle (NCP) at an overall resolution of 4.4 [A] with resolutions ranging from 4-6 [A] in Pol II and 6-8 [A] in the NCP. Pol II and the NCP adopt a defined orientation that could not be predicted from modelling. Pol II contacts DNA of the incoming NCP on both sides of the nucleosomal dyad with its domains clamp head and lobe. Comparison of the Pol II-NCP structure to known structures of Pol II complexes reveals that the elongation factors TFIIS, DSIF, NELF, PAF1 complex, and SPT6 can be accommodated on the Pol II surface in the presence of the oriented nucleosome. Further structural comparisons show that the chromatin remodelling enzyme Chd1, which is also required for efficient Pol II passage6,7, could bind the oriented nucleosome with its motor domain. The DNA-binding region of Chd1 must however be released from DNA when Pol II approaches the nucleosome, and based on published data8,9 this is predicted to stimulate Chd1 activity and to facilitate Pol II passage. Our results provide a starting point for a mechanistic analysis of chromatin transcription.

biochemistry↗

bioRxiv2018

Real-time cryo-EM data pre-processing with Warp

The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens is currently largely uncoupled from subsequent data evaluation, correction and processing. Therefore, the acquisition strategy is difficult to optimize during data collection, often leading to suboptimal microscope usage and disappointing results. Here we provide Warp, a software for real-time evaluation, correction, and processing of cryo-EM data during their acquisition. Warp evaluates and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. Warp also rapidly corrects micrographs for global and local motion, and estimates the local defocus with the use of novel algorithms. The software further includes a deep learning-based particle picking algorithm that rivals human accuracy to make the pre-processing pipeline truly automated. The output from Warp can be directly fed into established tools for particle classification and 3D image reconstruction. In a benchmarking study we show that Warp automatically processed a published cryo-EM data set for influenza virus hemagglutinin, leading to an improvement of the nominal resolution from 3.9 [A] to 3.2 [A]. Warp is easy to install, computationally inexpensive, and has an intuitive and streamlined user interface.

biophysics↗

bioRxiv2018

RNA polymerase II clustering through CTD phase separation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

molecular biology↗

bioRxiv2017

The interaction landscape between transcription factors and the nucleosome

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors (TFs) in regions that direct gene expression. However, the modes of interaction between TFs and nucleosomal DNA remain largely unknown. Here, we use nucleosome consecutive affinity-purification systematic evolution of ligands by exponential enrichment (NCAP-SELEX) to systematically explore interactions between the nucleosome and 220 TFs representing diverse structural families. Consistently with earlier observations, we find that the vast majority of TFs have less access to nucleosomal DNA than to free DNA. The motifs recovered from TFs bound to nucleosomal and free DNA are generally similar; however, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many TFs preferentially bind close to the end of nucleosomal DNA, or to periodic positions at its solvent-exposed side. TFs often also bind nucleosomal DNA in a particular orientation, because the nucleosome breaks the local rotational symmetry of DNA. Some TFs also specifically interact with DNA located at the dyad position where only one DNA gyre is wound, whereas other TFs prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals striking differences in TF binding to free and nucleosomal DNA, and uncovers a rich interaction landscape between the TFs and the nucleosome.

systems biology↗

bioRxiv2017

Loss of Wt1 in the murine spinal cord alters interneuron composition and locomotion

Rhythmic and patterned locomotion is driven by spinal cord neurons that form neuronal circuits, referred to as central pattern generators (CPGs). Recently, dI6 neurons were suggested to participate in the control of locomotion. The dI6 neurons can be subdivided into three populations, one of which expresses the Wilms tumor suppressor gene Wt1. However, the role that Wt1 exerts on these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed that these mice ran slower than controls with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0 and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and loss of Wt1 is associated with alterations in locomotion.

neuroscience↗