Detection of tetramerization domains in vivo by cooperative DNA binding to tandem λ operator sites

Mutational Analysis of the PapB Transcriptional Regulator inEscherichia coli

“…Based on plasmid pBF21, which carries the bacteriophage cI repressor gene under the control of a tandemly repeated lacUV5 promoter, and pBF22 in which the DNA encoding the oligomerization domain of the repressor (C-terminal domain) was deleted (14), we constructed plasmid pYN51 encoding the chimeric protein consisting of the N-terminal DNA binding domain of the cI repressor and the wt-PapB molecule (Table I). The test relies on the capability of an oligomerization-proficient protein domain to functionally replace the natural C-terminal domain of phage cI repressor conferring biological activity to the N-terminal DNA binding domain of the same repressor (21) (Fig. 5A).…”

Regulatory Interactions among Adhesin Gene Systems of Uropathogenic Escherichia coli

“…This assay is based on hybrid gene construction with the bacteriophage lambda cI repressor gene. It relies on the ability of the oligomerizationproficient protein domain to functionally replace the C-terminal domain of the phage repressor protein and thereby confer activity to the N-terminal DNA binding domain (57). Plasmids encoding different domains or hybrids were introduced into strain JH607, which can be used as a reporter system for cooperative binding of repressor proteins to adjacent operators (4).…”

Independent and Interchangeable Multimerization Domains of the AbrB, Abh, and SpoVT Global Regulatory Proteins

“…The in vivo assay developed by James Hu and colleagues for detection of protein domains having multimerization activity was used in this study. Methods for constructing and assaying the fusions were according to previously published protocols and information supplied by the Hu Laboratory as part of their generous package of vectors, strains, and phages required for the technique (15,32,33). The principle of the assay is that the DNA-binding N-terminal domain (DBD) of the cI repressor is unable to bind its cognate operator sequences unless fused to a peptide segment that can self-associate to form dimers or higher-order multimeric complexes.…”

“…To accomplish this, comparative ␤-galactosidase assays were performed using JH607 and XZ980 reporter strains harboring the fusion constructs. The properties of the cI-repressible operators present upstream of lacZ reporters in these phage have been previously described (2,15,33). Briefly, any cI-DBD fusion construct capable of dimerization will lead to repression of these reporters, but in the case of the JH607 reporter the formation of trimers or higher will give a greater If the repressed activity of the JH607 reporter is equal to or greater than the XZ980 reporter then the dimer state is indicated; a repressed activity in JH607 lower than that in XZ980 indicates formation of an oligomer state greater than dimerization.…”