Structural Characterization of Native RNA Polymerase II Transcription Complexes and Nucleosomes in Drosophila melanogaster

Abstract

Structural studies of eukaryotic RNA polymerase II (Pol II) transcription complexes often depend on in vitro assembly by mixing purified Pol II with synthetic DNA/RNA scaffolds, recombinant transcription factors, and/or histones, followed by stalling transcription at defined positions by adding selected nucleotide triphosphate substrates. These studies have yielded remarkable results for understanding nucleosome transcription by Pol II with elongation factors but may fail to represent transcription in native conditions. To investigate Pol II transcription within metazoan cells, we developed an approach to isolate the native transcription complexes from Drosophila melanogaster embryos. Utilizing one-step FLAG-tag affinity purification and mild chromatin treatment with Micrococcal Nuclease (MNase), we preserved the native transcription complex for cryo-EM and proteomics studies. In silico purification through the cryo-EM classifications determined structures of multiple forms of native transcription complex, nucleosome and other macromolecules. Remarkably, we determined the structures of metazoan Rpb4/Rpb7 stalk-less elongation complex as well as hexameric nucleosome lacking an H2A/H2B dimer, revealing that diverse elongation complexes and nucleosomes are involved in active transcription in vivo. Nucleosome is positioned only downstream of Pol II in the nucleosome elongation complex, underscoring it as a major energy barrier and a time-consuming step during Pol II progression through nucleosomal DNA. Proteomics identified co-purified factors responsible for initiation and elongation stages of transcription, as well as RNA modification factors. This study lays the groundwork for structural study of native transcription in eukaryotes, with future work focused on studies of transient and minor populations of transcription complexes.