Low-salt crystallization of T7 RNA polymerase: a first step towards the transcription bubble complex

DNA-dependent RNA polymerase is the key enzyme responsible for the biosynth esis of RNA, a process known as transcription. This process, which decodes the genetic information from DNA, is one of the most significant events in a biological system. The crystallization of both native and a chimeric T7/T 3 RNAP using high-salt conditions has been reported previously but these co nditions proved unsuitable for DNA-RNAP complex formation since at high-sal t concentrations the DNA binding affinity to RNAP is reduced. A search for low-salt crystallization conditions has yielded new low-salt crystals of na tive T7-RNAP, a chimeric T7-RNAP (T7/T3 RNAP) which contains the T3 promote r recognition sequence, and a T7-RNAP containing an N-terminal histidine ta g. The crystals, which are better suited for DNA-RNAP complex formation, be long to space group P3(1)21 with a = 136, c = 156 Angstrom, contain a singl e molecule per asymmetric unit and diffract to 2.7 Angstrom resolution. Pac king analysis shows that the new low-salt crystals have packing contacts si milar to those observed in the high-salt T7-RNAP crystals reported previous ly. The diffraction anisotropicity observed in crystals of T7 RNAP is expla ined in term of crystal packing.