Intragenic localization of amber and temperature-sensitive rpoD mutations affecting RNA polymerase sigma factor of Escherichia coli.

A set of multi-copy plasmids carrying part or all of the rpoD gene of Escherichia coli have been used to localize several amber and temperature-sensitive mutations affecting RNA polymerase sigma factor. In contrast to the plasmid carrying a complete rpoD sequence, plasmids carrying part of rpoD could not complement any of the rpoD mutations tested. However, the mutants harboring some of the latter plasmids produced wild-type recombinants at high frequency, provided that they carry the recA+ gene. The results permitted us to localize the rpoD mutations into one of the three intragenic segments. Thus, the rpoD285(ts) mutation (Harris et al. 1978) was located on the 0.8 kilobase segment defined by the Bg/II and BamHI sites in the middle segment, and the amber mutation rpoD40 (Osawa and Yura 1980) in the N-terminal 0.8 kilobase segment. Four additional amber mutations were also identified and classified into one of the segments of rpoD. It was further revealed that plasmids carrying certain amber mutations (rpoD47 or rpoD63) in the C-terminal segment of rpoD render all rpoD mutants tested (including rpoD47 and rpoD63 mutants themselves) able to grow at the restrictive condition. It is suggested that the enhanced synthesis of incomplete sigma polypeptides encoded by rpoD47 or rpoD63 phenotypically suppresses the defects in sigma function at least partially.