Targeting the Escherichia coli lac repressor to the mammalian cell nucleus.
We have previously shown that about 90% of total Escherichia coli lac repressor synthesized in mammalian cells is located in the cytoplasm [Hu and Davidson, Cell 48 (1987) 555-566]. To target a functional lac repressor to the nucleus, we mutated 10 nucleotides at the 3' end of the coding sequence, thus adding the nuclear localization signal of the simian virus 40 large-T antigen to the C terminus of the repressor. The mutant lacI gene and the wild-type (wt) gene, both in standard animal cell expression vectors, driven by the promoter of the Rous sarcoma virus long terminal repeat, were stably transfected into three rodent cell lines. In confirmation of our previous results, only about 10% of the wt repressor, but all of the mutant protein, was localized in the nucleus. DNase I footprint analyses showed that the mutant repressor retained the same operator DNA-binding specificity as wt repressor. Furthermore, both repressor-operator complexes could be dissociated by addition of isopropyl-beta-D-thiogalactopyranoside in vitro. However, the ratio of number of repressor molecules per nucleus that, by in vitro assay, could bind to the operator sequence to the number of monomer repressor polypeptides per nucleus, as determined by Western blotting, was about 1:4 for the wt repressor and about 1:30 for the mutant repressor. This suggests that: (a) the mutant repressor assembles into tetramers inefficiently; and/or (b) it has reduced binding affinity to the operator sequence; and/or (c) it has higher binding affinity to nonspecific DNA.
Hu
M C
MC
Division of Biology, California Institute of Technology, Pasadena 91125.
Davidson
N
N
D000957:Antigens, Viral, Tumor / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D000957:Antigens, Viral, Tumor; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside