MUTATIONAL ANALYSIS OF THE PUTATIVE NUCLEIC ACID-BINDING SURFACE OF THE COLD-SHOCK DOMAIN, CSPB, REVEALED AN ESSENTIAL ROLE OF AROMATIC ANDBASIC RESIDUES IN BINDING OF SINGLE-STRANDED-DNA CONTAINING THE Y-BOXMOTIF

The major cold-shock protein of Bacillus subtilis, CspB, is a member o f a protein family widespread among prokaryotes and eukaryotes that sh are the highly conserved cold-shock domain (CSD). The CSD domain Is in volved in transcriptional and translational regulation and was shown t o bind the Y-box motif, a cis-element that contains the core sequence ATTGG, with high affinity. The three-dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD d omain creates a surface rich in aromatic and basic amino acids that ma y act as the nucleic acid-binding site. We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site-directed mutagenesis. In gel retardation and ultraviolet cros s-linking experiments, the ability of CspB mutants to bind single-stra nded oligonucleotides (ssDNA) that contain the Y-box motif was investi gated. Single substitutions of three highly conserved phenylalanine re sidues (Phe-15, Phe-17, Phe-27) by alanine and substitution of one his tidine (His-29) by glutamine, all located within the putative RNA-bind ing sites RNP-1 and RNP-2, abolished the nucleic acid-binding activity of CspB. Conservative substitutions of Phe-15 to tyrosine (F15Y) show ed a small increase in binding affinity, whereas separate replacement of Phe-17 and Phe-27 by tyrosine caused a reduction in binding activit y. These and other substitutions including the conserved basic residue s Lys-7, Lys-13 and Arg-56 as well as the aromatic residues Trp-8 and Phe-30 strongly suggest that CspB uses the sidechains of these amino a cids for specific interaction with nucleic acids. Ultraviolet cross-li nking experiments for CspB mutants with ssDNA supported the idea of sp ecific CspB/nucleic acid interaction and indicated an essential role f or the aromatic and basic residues in this binding. In addition, two-d imensional nuclear magnetic resonance studies with F17A, K13Q, F15Y an d F27Y revealed that the mutants have the same overall structure as th e wild-type CspB protein.