Gene machines

Abstract:

A "bendy" protein sensor: A DNA-based sensor that uses folded DNA (through DNA kinks) and protein-induced bending to detect DNA-binding proteins is presented. Single-molecule sensing of a transcriptional activator (catabolite activator protein, CAP, which bends its DNA site by 80°) is demonstrated in solution and on surfaces, both in buffers and in cell lysates. The method should allow detection of a wide range of DNA-bending proteins.

Abstract:

λ-Repressor-operator sites interaction, particularly O(R)1 and O(R)2, is a key component of the λ-genetic switch. FRET from the dansyl bound to the C-terminal domain of the protein, to the intercalated EtBr in the operator DNA indicates that the structure of the protein is more compact in the O(R)2 complex than in the O(R)1 complex. Fluorescence anisotropy reveals enhanced flexibility of the C-terminal domain of the repressor at fast timescales after complex formation with O(R)1. In contrast, O(R)2 bound repressor shows no significant enhancement of protein dynamics at these timescales. These differences are shown to be important for correct protein-protein interactions. Altered protein dynamics upon specific DNA sequence recognition may play important roles in assembly of regulatory proteins at the correct positions.