Novel application of sRNA: stimulation of ribosomal frameshifting.

Small RNAs play an important role in regulation of gene expression in eukaryotic and eubacterial cells by modulating gene expression both at the level of transcription and translation. Here, we show that short complementary RNAs can also affect gene expression by stimulating ribosomal frameshifting in vitro. This finding has important implications for understanding the process of ribosomal frameshifting and for the potential application of small RNAs in the treatment of diseases that are due to frameshift mutations.

Prediction of proton chemical shifts in RNA. Their use in structure refinement and validation.

An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct., 76, 81-92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341-346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable analysis could be made of the structural dependencies of the 1H chemical shifts revealing their physical origin. For example, a down-field shift of either H2' or H3' or both indicates a high-syn/syn chi-angle. In an A-helix it is essentially the 5'-neighbor that affects the chemical shifts of H5, H6 and H8 protons. The H5, H6 and H8 resonances can therefore be assigned in an A-helix on the basis of their observed chemical shifts. In general, the chemical shifts were found to be quite sensitive to structural changes. We therefore propose that a comparison between calculated and observed 1H chemical shifts is a good tool for validation and refinement of structures derived from NOEs and J-couplings.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

Recognition properties of V3-specific antibodies to V3 loop peptides derived from HIV-1 gp120 presented in multiple conformations.

To identify structural constraints and amino acid sequences important for antibody recognition of the third variable domain (V3) of HIV-1 gp120, we have studied the solution conformation of three 35-mer circular V3 loop peptides derived from HIV-1 strains which differ in syncytium- (SI) and non-syncytium-inducing (NSI) capacity. In addition to 2D NMR and CD analyses, fluid- and solid-phase immunoassays were performed using V3-specific antibodies to V3 peptides and gp120 derived from different strains of HIV-1. NMR and CD spectroscopy indicated that circular and linear V3 loops exist in water as a dynamic ensemble of multiple conformations. Amino acid substitutions and biochemical modifications of the V3 loop were found to affect antibody binding depending on the presentation of the antigens. From NMR observations and immunological experiments, we provide evidence for a V3 loop specific monoclonal antibody interaction which is directed predominantly against linear epitopes rather than against discontinuous epitopes. The absence of a single defined solution conformation of 35-mer circular V3 peptides should be taken into account when using V3-related peptides to investigate structural elements in the V3 domain of the gp120 envelope protein of HIV-1 involved in biological processes of the virus.

Proton MR spectroscopy of wild-type and creatine kinase deficient mouse skeletal muscle: dipole-dipole coupling effects and post-mortem changes.

Localized proton MR spectra of mouse skeletal muscle obtained at 7 T show dipole-dipole coupling effects for creatine and putative taurine resonances and for the lactate methine signal. These effects are independent of the presence of creatine kinase. The intensity of the methylene (1)H resonance of creatine is not different between wild-type and creatine kinase deficient mice, which have a lower phosphocreatine content. (1)H-MR spectra acquired post-mortem from wild-type mouse skeletal muscle parallel to B(0) show a linewidth decrease for the methyl resonance of creatine and a 20% signal intensity loss for its methylene peak concurrent with the total breakdown of phosphocreatine as observed by (31)P-MR spectroscopy. However, with the muscle at the magic angle no changes in the appearance and intensity of creatine (and taurine) resonances are observed. These results indicate that the changes observed for creatine resonances are related to altered dipolar couplings and that the intensity of the methylene peak does not necessarily reflect muscular phosphocreatine content.

Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site.

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.

Structure and mechanism of formation of the H-y5 isomer of an intramolecular DNA triple helix.

H-DNA, thought to play a regulatory role in transcription, exists in two isomeric forms, H-y3 and H-y5. We present the first solution structure of a DNA fragment representing the H-y5 fold. The structure shows the H-y5 triple helix, and for the first time how in an H-DNA isomer the purine strand extension interacts with the triplex loop. It gives direct insight into the mechanism of H-DNA formation, and explains a host of biochemical and biophysical data on the relative stability of the H-DNA isomers. In addition, the observed interaction of the purine strand extension and the triplex loop provides new clues to the design of clamp-type triple helix-forming oligonucleotides.

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase.

The fluorescence signal of the single tryptophan residue (Trp69) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp69 in cutinase is caused by the breaking of the disulfide bond between Cys31 and Cys109 upon irradiation, while the amide-aromatic hydrogen bond between Ala32 and Trp69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.

NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors.

The backbone dynamics of Fusarium solani pisi cutinase in complex with a phosphonate inhibitor has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The results have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding the inhibitor. While the active site Ser120 is rigidly attached to the stable alpha/beta core of the protein, the remainder of the binding site is very flexible in the free enzyme. The other two active site residues Asp175 and His188 as well as the oxyanion hole residues Ser42 and Gln121 are only restrained into their proper positions upon binding of the substrate-like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our results are in contrast with the X-ray analysis results, namely that in the protein crystal, free cutinase has a well-defined active site and a preformed oxyanion hole and that it does not need any rearrangements to bind its substrate. Our solution studies show that cutinase does need conformational rearrangements to bind its substrate, which may form the rate-limiting step in catalysis.

Backbone dynamics of Fusarium solani pisi cutinase probed by nuclear magnetic resonance: the lack of interfacial activation revisited.

The backbone dynamics of Fusarium solani pisi cutinase has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The core of cutinase appears to be highly rigid. The binding site, including the oxyanion hole, is mobile on the microsecond to millisecond time scale, in contrast to the well-defined active site and preformed oxyanion hole elucidated by X-ray crystallography [Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G., and Cambillau, C. (1992) Nature 356, 615-618]. In this crystal structure, cutinase has a rather open conformation, in which the hydrophobic binding site is exposed. The observed mobility in solution most likely represents the interconversion between open and more closed conformations, like in a true lipase. The opening and closing motions are on a time scale which corresponds with the kinetics of the hydrolysis reaction, i.e., the millisecond range, which suggests that these conformational rearrangements form the rate-limiting step in catalysis. We conclude that the crystal structure probably represents one of the multiple conformations present in solution, which fortuitously is the active conformation. The implications of our findings are discussed with particular reference to the explanation of the lack of interfacial activation as found for cutinase.

Functionally important correlated motions in the single-stranded DNA-binding protein encoded by filamentous phage Pf3.

To elucidate the interplay between different parts of dimeric single-stranded DNA-binding proteins we have studied the correlated motions in the protein encoded by filamentous phage Pf3 via the combined use of 15N-NMR relaxation experiments, molecular dynamics simulations and essential dynamics calculations. These studies provide insight into the mechanism underlying the protein-DNA binding reaction. The most important motions can be described by a few essential modes. Most outstanding is the correlated symmetric motion of the DNA-binding wings, which are far apart in the structure. This motion determines the access of DNA to the DNA-binding domain. A correlation between the motion of the DNA-binding wing and the complex loop is indicated to play a role in the cooperative binding of the protein to DNA. These motions are in the nanosecond regime in correspondence with the 15N-NMR relaxation experiments.

Structure of the 3'-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.

Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Solution structure of the M13 major coat protein in detergent micelles: a basis for a model of phage assembly involving specific residues.

The three-dimensional structure of the major coat protein of bacteriophage M13, solubilized in detergent micelles, has been determined using heteronuclear multidimensional NMR and restrained molecular dynamics. The protein consists of two alpha-helices, running from residues 8 to 16 and 25 to 45, respectively. These two helices are connected by a flexible and distorted helical hinge region. The structural properties of the coat protein make it resemble a flail, in which the hydrophobic helix (residues 25 to 45) is the handle and the other, amphipathic, helix the swingle. In this metaphor, the hinge region is the connecting piece of leather. The mobility of the residues in the hinge region is likely to enable a smooth transformation from the membrane-bound form, mimicked by the structure in detergent micelles, into the structure in the mature phage. A specific distribution of the residues over the surface of the two helices was observed in the presented high-resolution structure of the membrane-bound form of the major coat protein as well as in the structure in the mature phage. All data suggest that this arrangement of residues is important for the interactions of the protein with the membrane, for correct protein-DNA and protein-protein interactions in the phage and for a proper growth of the phage during the assembly process. By combining our findings with earlier NMR results on the major coat protein in detergent micelles, we were able to construct a model that addresses the role of specific residues in the assembly process.

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Essential spaces defined by NMR structure ensembles and molecular dynamics simulation show significant overlap.

Large concerted motions of proteins which span its "essential space," are an important component of protein dynamics. We investigate to what extent structure ensembles generated with standard structure calculation techniques such as simulated annealing can capture these motions by comparing them to long-time molecular dynamics (MD) trajectories. The motions are analyzed by principal component analysis and compared using inner products of eigenvectors of the respective covariance matrices. Two very different systems are studied, the beta-spectrin PH domain and the single-stranded DNA binding protein (ssDBP) from the filamentous phage Pf3. A comparison of the ensembles from NMR and MD shows significant overlap of the essential spaces, which in the case of ssDBP is extraordinarily high. The influence of variations in the specifications of distance restraints is investigated. We also study the influence of the selection criterion for the final structure ensemble on the definition of mobility. The results suggest a modified criterion that improves conformational sampling in terms of amplitudes of correlated motion.