Selection for Tn10 Tet Repressor Binding to Tet Operator in Escherichia Coli: Isolation of Temperature-Sensitive Mutants and Combinatorial Mutagenesis in the DNA Binding Motif
AUTOR(ES)
Wissmann, A.
RESUMO
We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the β-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1204461Documentos Relacionados
- Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants.
- Tn10 tet operator mutations affecting Tet repressor recognition.
- Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor.
- Localized Mutagenesis for the Isolation of Temperature-Sensitive Mutants of ESCHERICHIA COLI Affected in Protein Synthesis
- Selection of Temperature-sensitive Activating Enzyme Mutants in Escherichia coli