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The role of the variable region in Tet repressor for inducibility by tetracycline.

TitleThe role of the variable region in Tet repressor for inducibility by tetracycline.
Publication TypeJournal Article
Year of Publication1997
AuthorsBerens C, Schnappinger D, Hillen W
JournalJ Biol Chem
Volume272
Issue11
Pagination6936-42
Date Published1997 Mar 14
ISSN0021-9258
KeywordsAnti-Bacterial Agents, Bacterial Proteins, Gene Deletion, Gene Expression Regulation, Bacterial, Mutagenesis, Site-Directed, Point Mutation, Repressor Proteins, Tetracycline
Abstract

A set of deletions and substitutions to alanine was introduced into the loop separating helices alpha8 and alpha9 of Tn10 Tet repressor (TetR). This region appears as an unstructured loop in the crystal structure of the TetR(D).([Mg-tc]+)2 complex and is the only internal segment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants shows that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected. All deletions have an induction-deficient TetRS phenotype, but the corresponding substitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates with their lack of inducibility. The association rate of [Mg-tc]+ to the TetR mutants is enhanced. Since none of the mutated residues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of TetR. We propose that the deletions in the loop shift the equilibrium between both forms toward the open, operator binding conformation.

DOI10.1074/jbc.272.11.6936
Alternate JournalJ Biol Chem
PubMed ID9054381

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