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

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Sep 1;68(Pt 9):1134-8. doi: 10.1107/S1744309112033507. Epub 2012 Aug 31.

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].

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallization
  • DNA / chemistry*
  • DNA-Directed RNA Polymerases / chemistry*
  • Potassium Chloride / chemistry
  • Sulfolobus / enzymology*

Substances

  • Potassium Chloride
  • DNA
  • DNA-Directed RNA Polymerases