Structure of the yeast SR protein Npl3 and Interaction with mRNA 3'-end processing signals.

Yeast Npl3 is homologous to SR proteins in higher eukaryotes, a family of RNA-binding proteins that have multiple essential roles in RNA metabolism. This protein competes with 3'-end processing factors for binding to the nascent RNA, protecting the transcript from premature termination and coordinating transcription termination and the packaging of the fully processed transcript for export. The NMR structure of its RNA-binding domain shows two unusually compact RNA recognition motifs (RRMs), and identifies the RNA recognition surface in Npl3. Biochemical and NMR studies identify a class of G+U-rich RNA sequences with high specificity for this protein. The protein binds to RNA and forms a single globular structure, but the two RRMs of Npl3 are not equivalent, with the second domain forming much stronger interactions with G+U-rich RNA sequences that occur independently of the interaction of the first RRM. The specific binding to G+U-rich RNAs observed for the two RRMs of Npl3 is masked in the full-length protein by a much stronger but non-sequence-specific RNA-binding activity residing outside of its RRMs. The preference of Npl3 for G+U-rich sequences supports the model for its function in regulating recognition of 3'-end processing sites through competition with the Rna15 (yeast analog of human CstF-64 protein) subunit of the processing complex.

The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection.

To illuminate the evolutionary pressure acting on the folding free energy landscapes of naturally occurring proteins, we have systematically characterized the folding free energy landscape of Top7, a computationally designed protein lacking an evolutionary history. Stopped-flow kinetics, circular dichroism, and NMR experiments reveal that there are at least three distinct phases in the folding of Top7, that a nonnative conformation is stable at equilibrium, and that multiple fragments of Top7 are stable in isolation. These results indicate that the folding of Top7 is significantly less cooperative than the folding of similarly sized naturally occurring proteins, suggesting that the cooperative folding and smooth free energy landscapes observed for small naturally occurring proteins are not general properties of polypeptide chains that fold to unique stable structures but are instead a product of natural selection.

Decoding RNA motional codes.

When proteins and small molecules bind to RNA, they often alter its conformation. These structural changes are an essential aspect of the ability of RNA to sense signaling molecules and modulate gene expression. Thus far, few studies have been dedicated to understanding how RNA moves at a residue level and how these motions change upon complex formation. A recent report highlights how intrinsic motions in RNA correlate with its ability to bind to cognate ligands.

Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR.

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 A decrease in the separation between phosphorothioate and 2'F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR-tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P-19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA-peptide complex.

Protein and RNA dynamics play key roles in determining the specific recognition of GU-rich polyadenylation regulatory elements by human Cstf-64 protein.

The N-terminal domain of the 64 kDa subunit of the cleavage stimulation factor (CstF-64) recognizes GU-rich elements within the 3'-untranslated region of eukaryotic mRNAs. This interaction is essential for mRNA 3' end processing and transcription termination, and its strength affects the efficiency of utilization of different polyadenylation sites. The structure of the RNA-binding N-terminal domain of CstF-64 showed how the N-terminal RNA recognition motif of CstF-64 recognizes GU-rich RNAs. However, it is still perplexing how this protein can bind selectively to RNAs that are rich in G and U residues regardless of their detailed sequence composition, yet discriminate effectively against non-GU-RNAs. We investigated by NMR the dynamics of the CstF-64 RNA-binding domain, both free and bound to two GU-rich RNA sequences that represent polyadenylation regulatory elements. While the free protein displays the motional properties typical of a well-folded protein domain and is uniformly rigid, the protein-RNA interface acquires significant mobility on the micro- to millisecond time-scale once GU-rich RNAs binds to it. These motional features, we propose, are intrinsic to the functional requirement to bind all GU-rich sequences and yet to discriminate against non-GU-rich RNAs. This behavior may be a general mechanism by which some RNA-binding proteins are able to bind to classes of sequences, as opposed to a well-defined sequence or consensus.