The structure of Rap30-DBD was solved by multi-nuclear NMR, revealing some of the structural relationship between components of the prokaryotic and eukaryotic RNA polymerase complex. (Caroline M. Groft, Sacha N. Uljon, Rong Wang, and Milton H. Werner (1998) PNAS USA 95, 10872-10877). The analogy between prokaryotes and eukaryotes has been extended to construct a chimera between E coli sigma70 and Rap30 in order to overcome solubility problems in the former which have precluded structural analysis. This has now been accomplished and highly soluble form of sigma70 has been produced and is presently being screened for activity. The replacement of region 4 of sigma70 with the Rap30-DBD that resulted in a soluble form of sigma70 suggests that the determinants of solubility of sigma70 are likely to reside in the 60 amino acids comprising region 4. Mutagenesis/mass spec analysis is under way to prepare mutants of sigma70 that are inherently soluble and retain native activity.
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