The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin ␣ through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS (ϳ3 kcal/mol) as well as the energy of inhibition of the importin ␣ importin ␤-binding domain (ϳ3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin ␣-importin ␤ complex. On this scale, a functional NLS has a binding constant of ϳ10 nM, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 M. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.The sequestering of genetic material in the nucleus by eukaryotic cells provides a powerful mechanism for the regulation of gene expression and other cellular processes through the selective translocation of proteins between the nucleus and the cytoplasm (1-3). Recently, the regulated transport of proteins across the nuclear envelope has been recognized as a crucial step in an increasing number of cellular processes (4 -6). Understanding the mechanisms of regulated protein translocation through nuclear pores requires a detailed definition of the signals that mark a macromolecular complex for nuclear import or export.The best characterized mechanism for translocation across the nuclear envelope is protein import which depends on the "classical" nuclear localization signal (NLS) 1 (7). This NLS consists of a cluster of basic residues (monopartite) or two clusters of basic residues separated by 10 -12 residues (bipartite) (8,9). This signal is recognized by the heterodimeric import receptor complex comprising importin ␣ and importin ␤ (3). Importin ␣ is an adapter protein that consists of a small N-terminal importin ␤-binding (IBB) domain and a larger Cterminal NLS-binding domain (10 -14). Importin ␤ does not directly interact with the NLS cargo but acts to direct importin ␣ to the nuclear pore (15, 16). In the absence of importin ␤, "NLS-like" sequences of the N-terminal IBB domain form an intramolecular bond with the NLS-binding site inhibiting the interaction between importin ␣ and the NLS cargo. Evidence for this auto-inhibition is found in the crystal structure of full-length importin ␣ as well as in vitro binding assays (16 -19). Thus, the interaction between importin ␣ and the NLS cargo is regulated by importin ␤. In an analogous m...

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Sequence-nonspecific binding of RNA, recognition of a 7-methylguanosine 5' mRNA cap, and methylation of a nucleic acid backbone are three crucial and ubiquitous events in eukaryotic nucleic acid processing and function. These three events occur concurrently in the modification of vaccinia transcripts by the methyltransferase VP39. We report the crystal structure of a ternary complex comprising VP39, coenzyme product S-adenosylhomocysteine, and a 5' m7 G-capped, single-stranded RNA hexamer. This structure reveals a novel and general mechanism for sequence-non-specific recognition of the mRNA transcript in which the protein interacts solely with the sugar-phosphate backbone of a short, single-stranded RNA helix. This report represents the first direct and detailed view of a protein complexed with single-stranded RNA or 5'-capped mRNA.

VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap-specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 A crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis data suggest two possible RNA-binding sites with novel underlying architecture, one of which forms a cleft spanning the region adjacent to the methyltransferase active site. This report provides a prototypic structure for an RNA methyltransferase, a protein that interacts with the mRNA 5' cap, and an intact poxvirus protein.