Protein-directed reorganization of DNA underlies mechanisms of transcription, replication, and recombination. A molecular model for DNA reorganization in the regulation of gene expression is provided by the sequence-specific high-mobility-group (HMG) box. Structures of HMG-box complexes with DNA are characterized by expansion of the minor groove, sharp bending toward the major groove, and local unwinding of the double helix. The Raman vibrational signature of such DNA reorganization has been identified in a study of the SRY HMG box, encoded by the human male-determining region of the Y chromosome. We observe in the human SRY-HMG:DNA complex extraordinarily large perturbations to Raman bands associated with vibrational modes of the DNA backbone and accompanying large increases in intensities of Raman bands attributable to base unstacking. In contrast, DNA major-groove binding, as occurs for the bZIP protein GCN4 [Benevides, J. M., Li, T., Lu, X.-J., Srinivasan, A. R., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 548-556], perturbs the Raman signature of DNA only marginally. Raman markers of minor-groove recognition in the human SRY-HMG:DNA complex are due primarily to perturbation of specific vibrational modes of deoxyribose moieties and presumably reflect desolvation at the nonpolar interface of protein and DNA. These Raman markers may be diagnostic of protein-induced DNA bending and are proposed as a baseline for comparative analysis of mutations in SRY that cause human sex reversal.

A novel RNA recognition motif is characterized in an arginine-rich peptide. The motif, derived from lambda transcriptional antitermination protein N, regulates an RNA-directed genetic switch. Its characterization by multidimensional nuclear magnetic resonance (NMR) demonstrates specific RNA-dependent folding of N- and C-terminal recognition helices separated by a central bend. The biological importance of the bent alpha-helix is demonstrated by mutagenesis: binding is blocked by substitutions in the N peptide or its target (the boxB RNA hairpin) associated in vivo with loss of transcriptional antitermination activity. Although arginine side chains are essential, the peptide is also anchored to boxB by specific nonpolar contacts. An alanine in the N-terminal helix docks in the major groove of the RNA stem whereas a tryptophan in the C-terminal helix stacks against a purine in the RNA loop. At these positions all 19 possible amino acid substitutions have been constructed by peptide synthesis; each impairs binding to boxB. The pattern of allowed and disallowed substitutions is in accord with the results of random-cassette mutagenesis in vivo. The helix-bend-helix motif rationalizes genetic analysis of N-dependent transcriptional antitermination and extends the structural repertoire of arginine-rich domains observed among mammalian immunodeficiency viruses.

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SRY, an architectural transcription factor encoded by the sex-determining region of the Y chromosome, initiates testicular differentiation in mammalian embryogenesis. The protein contains a high-mobility group (HMG) box, a DNA-bending motif conserved among a broad class of nuclear proteins. Mutations causing human sex reversal (46, XY pure gonadal dysgenesis) are clustered in this domain. Basic N-and C-terminal regions of the HMG box are each proposed to provide nuclear localization signals. The significance of the Cterminal basic cluster (SRY residues 130 -134) is uncertain, however, as its activity in cell culture varies with assay conditions. To test its importance, we have investigated a C-terminal sex-reversal mutation (R133W, position 78 of the HMG box). This de novo mutation impairs nuclear localization but not specific DNA binding or sharp DNA bending. Correlation between these properties and the phenotype of the patient suggests that nuclear localization of SRY is required for testicular differentiation and directed in part by the C-terminal basic cluster. To our knowledge, these results provide the first example of impaired organogenesis due to a nuclear localization signal mutation.SRY, the testes-determining factor encoded by the human Y chromosome (1), contains a high-mobility group (HMG) 1 box (2-4), a conserved motif of DNA bending (Fig. 1, A and C, and Ref. 5). Mutations in SRY are associated with 46, XY pure gonadal dysgenesis leading to failure of testicular differentiation and female somatic phenotype (XY sex reversal; Refs. 3 and 6 -8). Clinical mutations cluster in the HMG box 2 and most commonly impair specific DNA binding (7,9,10). SRY is a nuclear protein (11) expressed in the primordial Sertoli cells of the differentiating gonadal ridge (12-14). Although SRY is presumed to function as an architectural transcription factor (9, 15, 16), its downstream genetic pathway is not well characterized (for a review, see Ref. 17).Immunohistochemical studies of murine and human embryos have demonstrated that SRY is a nuclear protein (11,18). Nuclear localization signals (NLSs) in human SRY have been defined in cell culture. Berta and colleagues (11), using microinjection of proteins in adult human fibroblastic cells, identified an NLS in the N-terminal region of the human HMG box 3 ( Fig. 2A; SRY residues 59 -75). This NLS comprises two sets of basic amino acids separated by 12 residues (Fig. 2B), features characteristic of bipartite NLS motifs in diverse proteins (19,20). An isolated N-terminal SRY peptide (residues 58 -78) was shown to be sufficient to direct nuclear translocation of coupled rabbit IgG (protein SRY21 in Fig. 2A). By contrast the remainder of the HMG box (residues 74 -137) was unable to direct nuclear translocation of coupled rabbit IgG (protein SRY64 in Fig. 2A). Although these findings appear to exclude a second NLS in SRY, Sü dbeck and Scherer (21) subsequently used a complementary methodology (transient transfection of SRY-␤-galactosidase fusion genes in COS-7 cells; Fig. 2B)...