1986 Volume 17 Issue 4 Pages 499-513
In Esherichia coli, most of cellular proteins are synthesized at constant rate through a cell cycle. However, there are a class of proteins such as β-galactosidase encoded by lacZ gene which are synthesized at a specific stage of the cycle. These cell-cycle dependent proteins are found in cytoplasm as well as outer and cell membrane. The previous studies have indicated that synthesis of these cell-cycle dependent proteins is regulated by both gene products of divE and dnaJ, although the precise mechanism is not determined. In order to obtain the molecular mechanism of the regulation, we tried to construct a fusion gene in which the promoter of cell-cycle dependent operon is replaced by that of cell-cycle independent one. The ptrp-lacZ fusion gene plasmid we constructed has regulator regions of tryptophan operon (cell-cycle independent) upstream of lacZ gene (cell-cycle dependent). We determined the nucleotide sequence of ptrp-lacZ and transduced it into bacteria to confirm that its hybrid β-galactosidase protein is synthesized under the control of tryptophan promoter. In the divE mutant which has a temperature sensitive defect of cell-cycle dependent protein synthesis, the chimeric β-galactosidase encoded by the hybrid gene was almost normally synthesized at the non-permissive temperature, in contrust to synthesis of intact β-galactosidase. We examined the in vitro DNA directed β-galactosidase synthesis using λ-dlac and ptrp-lacZ DNA as templates. It was found that the S-30 fraction obtained from the dnaJ mutant grown at permissive temperature had a normal level of β-galactosidase synthesizing activity wheares the S-30 fraction prepared from cells exposed to nonpermissive temperature lost its activity when λdlac DNA as a template. On the other hand, the synthesis of tryptophan promoter mediated chimeric β-galactosidase occurred at normal rates with both S-30 fractions. These results indicate that the temperature sensitive defect in divE and dnaJ mutants has a promoter specificity and resides on transcriptional step of protein synthesis.
