Sample Preparation of the Elongation Complex +39 for Cryo-Electron Microscopy

Transcription in the cell is performed by specialized enzymes (RNA polymerases). RNA polymerases transcribe DNA with the formation of elongation complexes (EC), including those involved in transcription regulation. Cryo-electron microscopy (cryo-EM) allows us to determine the structures of these complexes and clarify the mechanisms of chromatin transcription. However, the preparation of EC samples suitable for cryo-EM study presents certain difficulties. In this study, the development of protocols for preparing EC samples with the location of RNAP active center at the position +39 from the nucleosome entrance (EC+39) was carried out. The formation of complexes was confirmed by the methods of electrophoresis and negative stain electron microscopy. The developed methods can be used for studying EC+39 using cryo-EM method.