The role of rigidity in DNA looping-unlooping by AraC

Abstract

We applied two experiments useful in the study of ligandregulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli L-arabinose operon. In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites. The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences. Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains. The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose. When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains. These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected.

DNA twisting flexibility and the formation of sharply looped protein–DNA complexes

“…Such large-scale conformational changes are possible in principle but have not been documented. Indeed, the available evidence argues in favor of rigid, not flexible, regulatory protein monomers (50). Our findings establish that sharply looped DNA itself inherently has the flexibility needed to restore the protein-protein interactions at a low cost in free energy simply by under-or overtwisting the DNA to restore the total twist back to its original value (Fig.…”

“…If the protein were sufficiently flexible, then a large-scale protein conformational change could restore the protein-protein contacts (state IV). However, such hypothetical flexibility has not been documented, and the available evidence argues that it sometimes may not exist (50). Alternatively, under-or overtwisting the looped DNA (state III) restores the protein-protein interactions, with no requirement for flexibility in the protein.…”

Mutational Analysis of the Escherichia coli melR Gene Suggests a Two-State Concerted Model To Explain Transcriptional Activation and Repression in the Melibiose Operon

“…Hence, in the X-ray structure of the AraC N-terminal domain without arabinose, this arm is unstructured and cannot be seen. The light switch model proposes that, in the absence of arabinose, the interaction of the AraC N-terminal arm with the C-terminal DNA-binding domain constrains the subunits of the AraC dimer in an orientation that makes it energetically favorable to bind to distal (O2 and I1) rather than adjacent (I1 and I2) targets (11,25). This model is supported by genetic analyses, notably, mutations that alter amino acids in the AraC N-terminal arm that result in AraC-dependent activation of paraBAD in the absence of arabinose (19,21,22,34,35).…”

Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3

“…Under inducing conditions these promoter mutations would behave like a box 2 mutation, as the repressor is mainly bound to box 2 in that case. Even though we cannot totally exclude the possibility that two different proteins bind to the different operator sequences within the xlnC promoter-the first responding to substrate induction while the second is responsible for glucose repression-we propose, by analogy with what is known of the regulation of the araBCD operon in E. coli (Harmer et al 2001), that the negative regulatory effect of the specific repressor of xlnC relies, in the presence of glucose, on the formation of a strong repressing loop involving box 3 and box 1 or perhaps part of box 2. Upon addition of xylan, this repressing loop would be undone and the repressor binding site would be shifted to box 2 slightly farther upstream, thus allowing some xlnC expression.…”