Gene expression, transcriptional regulation and transcription-coupled repair (TCR) are processes critically dependent on large macromolecular complexes involving RNAPII. The size and complexity of these assemblies complicates the path toward mechanistic understanding. Herein, we have used a combined approach involving electron microscopy (EM) and computational modeling to shed light on these essential biological complexes.We recently developed models for human RNA polymerase II (RNAPII) transcription elongation complex stalled at a cisplatin DNA adduct. While structures of yeast RNAPII are available with and without the DNA/RNA hybrid and in multiple functional states, experimental difficulties have prevented structural biologists from obtaining high-resolution crystal structures for the transcription complexes of human RNA polymerase II. We took advantage of a recently solved high quality EM structure of hRNAPII (7-10 � resolution) and constructed an atomistic model of the hRNAPII elongation complex by molecular dynamics flexible fitting (MDFF). The model is the most detailed available for the human elongation complex and led us to highlight important differences with the yeast crystal structures. Furthermore, we were able to resolve the entire transcription bubble. Stalling of the actively transcribing polymerase at a lesion appears to trap the enzyme into a closed cleft conformation in which the clamp of RNAPII is stabilized. Extra densities absent in free RNAPII, corresponding to the path of upstream DNA and downstream DNA product, were clearly discernable, as well as flexible domains not resolved in the yeast structure and our model was able to capture all these features.

Integrative modeling of complex biological asssemblies in transcription

246th National Meeting (2013)

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