Abstract
The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.
Highlights
A large number of diverse DNA transactions require the cooperative formation of higherorder nucleoprotein complexes including transcription, replication, DNA repair, and DNA segregation
In this work we study the bacterial nucleoid-associated protein, Fis, and its association with a variety of DNA sites in order to investigate the role of DNA sequence in target selection and protein-stabilized DNA shape changes within the bound complexes
Fis is remarkable in its ability to bind with high affinity to DNA sites that are poorly related at the sequence level
Summary
A large number of diverse DNA transactions require the cooperative formation of higherorder nucleoprotein complexes including transcription, replication, DNA repair, and DNA segregation. In this work we study the bacterial nucleoid-associated protein, Fis, and its association with a variety of DNA sites in order to investigate the role of DNA sequence in target selection and protein-stabilized DNA shape changes within the bound complexes. Fis is one of a handful of nucleoid-associated proteins that bind prolifically throughout bacterial chromosomes. It is among the most abundant DNA binding proteins under rapid growth conditions in Enterobacteriaceae. Fis cooperatively recruits or competes with alternative regulators or effector proteins including recombinases In several of these reactions, Fis-induced changes in DNA shape has been shown to contribute to the formation of higher-order nucleoprotein complexes [5,14,15,16]
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