Hairpin loops are important structural elements of RNA, helping to define the three-dimensional structure of large RNAs and providing potential nucleation sites for RNA folding and interaction with other nucleic acids and proteins. Little, however, is known about the conformation of RNA hairpins, most of what we know coming from transfer RNA crystal structures and from studies of DNA hairpins. We report here the determination of the structure of a very stable and common RNA hairpin, 5'GGAC(UUCG)GUCC (loop nucleotides in parenthesis), by NMR spectroscopy. The sequence C(UUCG)G occurs very often in RNA and may be a nucleation site for RNA folding and a protein-binding site. A high-resolution structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR. The loop is stabilized by a G.U base pair, with guanine in the syn conformation, a cytosine-phosphate contact and extensive base stacking. These findings and other structural features of the loop can explain the unusual stability of the hairpin and suggest why reverse transcriptase cannot read through the loop, although it can transcribe through other kinds of RNA secondary structure.

The structure of a very common RNA hairpin, 5'GGAC(UUCG)GUCC, has been determined in solution by NMR spectroscopy. The loop sequence, UUCG, occurs exceptionally often in ribosomal and other RNAs, and may serve as a nucleation site for RNA folding and as a protein recognition site. Reverse transcriptase cannot read through this loop, although it normally transcribes RNA secondary structure motifs. A hairpin with that loop displays unusually high thermodynamic stability; its stability decreases when conserved nucleotides are mutated. The three-dimensional structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR using distance geometry, followed by restrained energy minimization. The structure was well-defined despite the conservative use of interproton distances, by constraining the backbone conformation by means of scalar coupling measurements. A mismatch G.U base pair, with syn-guanosine, closes the stem. This hairpin has a loop of only two nucleotides; both adopt C2'-endo sugar pucker. A sharp turn in the phosphodiester backbone is stabilized by a specific cytosine-phosphate contact, probably a hydrogen bond, and by stacking of the cytosine nucleotide on the G.U base pair. The structural features of the loop can explain the unusual thermodynamic stability of this hairpin and its sensitivity to mutations of loop nucleotides.

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (APT) or incorrect (G-T, COT, T-T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon disso- energies of dissociation of correct and incorrect base pairs account for nucleotide insertion fidelity? To address these questions, a thermodynamic analysis (3) is made of melting data for oligonucleotide duplexes containing matched and mismatched template-primer termini. The thermodynamic measurements are compared with enzyme kinetic data obtained with the same DNA sequences under the same conditions, for right and wrong nucleotide insertions (4), and for elongation from matched and mismatched template-primer termini. MATERIALS AND METHODSPurified Drosophila DNA polymerase a holoenzyme (5) was a generous gift of I. R. Lehman (Stanford University, Stanford, CA). Four versions of a 20-base DNA primer (5'-TGATATTCACAACGAATGGN-3'), where N = A, C, G, or T), complementary in sequence (except for terminal base N) to bases 2242-2222 in wild-type M13 DNA (6), were synthesized by conventional solid-phase methods. The template was single-stranded DNA isolated from wild-type M13 phage grown in Escherichia coli strain JM103. Each primer was labeled at the 5' end with 32P using [y-32P]ATP (4500 Ci/ mmol; 1 Ci = 37 GBq) purchased from ICN Radiochemicals and T4 polynucleotide kinase from United States Biochemicals, Cleveland, OH. Procedures for primer 5'-end-labeling and hybridizing to template were the same as described (4).Synthetic DNA duplexes used in melting experiments, representing the last 9 base pairs in the primer-template complexes and differing only in the terminal base pair (NOT), were prepared by annealing equimolar amounts of the component 9-base strands synthesized in the same way as primers.DNA Polymerase Reactions. To measure extension rates at primer 3' ends (N opposite T), with dTTP as substrate for addition of T opposite A, reactions were carried out in the same way with each of the four 5'-end-labeled primers hybridized to M13 template as illustrated in Fig. lb

The crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the middle of the sequence did not form an internal loop, but rather a highly regular double-helix incorporating the non-Watson-Crick base pairs, G.U and U.C. This is the first direct observation of a U.C (or T.C) base pair in a crystal structure. The U.C pairs each form only a single base-base hydrogen bond, but are stabilized by a water molecule which bridges between the ring nitrogens and by four waters in the major groove which link the bases and phosphates. The lack of distortion introduced in the double helix by the U.C mismatch may explain its low efficiency of repair in DNA. The G.U wobble pair is also stabilized by a minor-groove water which bridges between the unpaired guanine amino and the ribose hydroxyl of the uracil. This structure emphasizes the importance of specific hydrogen bonding between not only the nucleotide bases, but also the ribose hydroxyls, phosphate oxygens and tightly bound waters in stabilization of the intramolecular and intermolecular structures of double helical RNA.

A model for the solution structure of an RNA tetraplex, (rUGGGGU)4, has been obtained by two-dimensional NMR spectroscopy and molecular dynamics. The molecule is parallel stranded and Hoogsteen base-paired in 50 mM KCl, and it is so stable that three of its six imino protons have exchange half-lives measured in days at 40 degrees C. The tetraplex is stabilized by base stacking and by the hydrogen bonds in four G quartets and at least one U quartet. This is the first indication of the existence of U-quartet structures of which we are aware.

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.

In eukaryotic cells, apoptosis and cell cycle arrest by the Ras 3 RASSF 3 MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long ␣-helices (h2/h2 ) provides an elongated binding interface between the two monomers, and the short 3 10 helices (h1/h1 ) are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.tumor suppressor ͉ cell cycle arrest ͉ Hippo ͉ Salvador R ecent work in cellular homeostasis has uncovered a pathway mediated by the MST (mammalian sterile 20-like kinase) family, the human ortholog for Hippo (Hpo), which promotes apoptosis and restricts cell proliferation in conjunction with RASSF family tumor suppressors and/or the scaffold protein Salvador (Sav) (1-6). This pathway is characterized by a unique interaction motif called SARAH (for Sav/Rassf/Hpo), which connects the proteins involved in this pathway (7).Mammalian sterile 20-like kinase 1 (Mst1, also called STK4) is a member of a family of serine/threonine kinases that show similarity to Ste20, an upstream activator of the MAPK pathway in budding yeast (8,9). Mst1 is cleaved by caspase 3, which is triggered either by the activation of death receptors, such as Fas and the TNF-␣ receptor, or by exposure of the cells to inducers of apoptosis, such as staurosporine or ceramide (10-13). Whereas intact Mst1 is localized predominantly in the cytoplasm, the catalytic fragment of Mst1 generated by caspase-mediated cleavage translocates to the nucleus and phosphorylates histone H2B at Ser-14, resulting in chromatin condensation, DNA fragmentation, and, ultimately, cell death by apoptosis (14,15).A Drosophila homolog of Mst1/2, Hippo (Hpo), together with Salvador (Sav) and Warts (Wts), promotes both proper exit from the cell cycle and apoptosis during development (1-3). Mst1 and Mst2 have also been shown to associate with members of the RASSF family of tumor suppressors, such as Rassf1 and Rassf5 (also known as Nore1), all of which contain a conserved Rasassociation (RA) domain, with both of the MST and RASSF proteins colocalizing to microtubules throughout the cell cycle (4-6). Whereas purified recombinant Rassf1A inhibited the kinase activity of ...

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