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Combinatorial Redesign of the DNA Binding Specificity of a Prokaryotic Helix-Turn-Helix Repressor. Katja Fromknecht, 2003.Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences . Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences . Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a  bacteriophage-specific repressor were identified . The libraries contained variations within the HTH sequence at only three positions . In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences . For the best 434 Cro variant identified, an apparent Kd for
OR3 of 1 nM was observed . In competition experiments, this Cro variant was observed to be highly selective . We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix . Important characteristics of the screening system responsible for the successful identifications are discussed . Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes .
YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System. Petra J. Edqvist, 2003.Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells . At 37°C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface . Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol . Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops . Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion . These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system . Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation . These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis .
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