Soaking of DNA into crystals of archaeal RNA polymerase achieved by desalting in droplets

Abstract

Transcription is a fundamental process across the three domains of life and is carried out by multi-subunit enzymatic DNA-directed RNA polymerases (RNAPs). The interaction of RNAP with nucleic acids is tightly controlled for precise and processive RNA synthesis. Whilst a wealth of structural information has been gathered on the eukaryotic Pol II in complex with DNA/RNA, no information exists on its ancestral counterpart archaeal RNAP. Thus, in order to extend knowledge of the archaeal transcriptional apparatus, crystallization of Sulfolobus shibatae RNAP (molecular mass of ∼400 kDa) with DNA fragments was pursued. To achieve this goal, crystal growth was first optimized using a nanoseeding technique. An ad hoc soaking protocol was then put into place, which consisted of gently exchanging the high-salt buffer used for apo-RNAP crystal growth into a low-salt buffer necessary for DNA binding to RNAP. Of the various crystals screened, one diffracted to 4.3 Å resolution and structural analysis showed the presence of bound DNA [Wojtas et al. (2012). Nucleic Acids Res. 40, doi:10.1093/nar/gks692]. © 2012 International Union of Crystallography.