Dna Looping By Lactose Repressor Requires Tetramer Opening

Abstract

Transcription of the bacterial genes involved in lactose metabolism is controlled by lactose repressor protein (LacI). LacI can bind simultaneously to two operators, forming a loop on the intervening DNA. Looping is essential for efficient repression, as demonstrated by the effects of deletion of the auxiliary operators. The protein is a dimer of dimers: in the crystal structure of LacI, the two dimers are arranged in a V-shape, and each dimer binds a DNA operator. Recently, theoretical and experimental lines of evidence have suggested various possible loop structures associated with different LacI tetramer conformations (adopted by varying the inter-dimer angle through flexion at the C-terminal tetramerization domain). Different DNA binding topologies can also contribute to the complexity of available protein/DNA conformations. We employed the single-molecule tethered particle motion (TPM) method, in combination with chemical crosslinking of LacI protein mutants, to specifically address the role of tetramer opening in loop formation. Measurements on the wild-type and mutant LacI variants, with native cysteines removed and single cysteines placed at selected sites, confirmed previous observations of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein to various degrees by chemical crosslinking of the introduced cysteines with reagents of different spacer-arm lengths induces pronounced effects. Crosslinking the dimers at residue 36 (in the N-terminal DNA binding domain) completely suppresses looping (with no effect on binding to 40 bp operator DNA). Crosslinking at position 231 (near the C-terminal tetramerization domain) changes the looping geometry as detected by TPM. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations in which the protein clearly must adopt a structure very different from the classic crystallographic V-shape.