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Biomedical subjects

G Varani

Publications and source records attributed to G Varani.

At least 55 records · Page 3Linked to original sources
PubMed1996

Structure of the polyadenylation regulatory element of the human U1A pre-mRNA 3'-untranslated region and interaction with the U1A protein.

The N-terminal RNP domain of U1A binds two different RNA substrates with high affinity and specificity: stem-loop II of the U1 snRNA and a complex secondary structure in the 3'-untranslated region (3'-UTR) of the U1A pre-mRNA. Both RNAs contain a single-stranded sequence which is the main site of interaction with the protein, but in completely different structural contexts. Here we describe the solution structure of the free 3'-UTR RNA molecule and the NMR characterization of its complex with the U1A protein N-terminal domain. The structure of the free RNA indicates that the stems are nearly canonical A-form helices and that the single-stranded region contains local stacking interactions in the context of a generally flexible structure. Upon protein binding, the internal loop region folds into an ordered structure containing significant changes in the local stacking interactions. These results demonstrate the role of RNA structure and folding in specific RNA-protein recognition.

Humans↗
PubMed1995

The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5' leader sequence found on all HIV-1 mRNAs. We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity. In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure. The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints. The U23 residue at the 5' end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR. U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue. However, the interaction of this guanidinium group with TAR is not the only source of binding specificity. Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR, suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat. These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction.

Amino Acid Sequence↗
PubMed1995

Structure of the P1 helix from group I self-splicing introns.

The upstream cleavage site of group I self-splicing introns is identified by an absolutely conserved U.G base-pair within a double helix. Mutant introns with a wobble C.A substitute are catalytically active, but all other combinations of nucleotides at these positions abolish splicing, suggesting that an unusual RNA structure generated by the wobble pair is recognized by the catalytic intron core. The solution structure of a 20-mer oligonucleotide containing a UUCG tetraloop hairpin and a U.G wobble pair within a double helix was determined by NMR spectroscopy without any assumptions on RNA conformation. Isotopically (15N/13C)-labelled RNA was used to collect an unusually large number of experimental constraints (703 in total, corresponding to approximately 35 constraints per nucleotide) leading to the determination of a structure with very high precision (overall root-mean-square-deviation (rmsd) between 20 converged structures 1.22 A, local rmsd 0.6 A for the tetraloop and 0.85 A for the stem). Analysis of the double helical structure at the conserved U.G wobble pair reveals local distortions from the regular A-form pattern, that may constitute the characteristic feature of U.G wobble pair recognized by the group I intron core and by amino acyl tRNA synthetases. Re-examination of the previously determined tetraloop structure reveals a novel U.G base-pair with a syn guanosine and hydrogen bonding contacts involving both base protons and a sugar 2'-OH. This explains the great stability of RNA UUCG loops when compared with DNA loops of identical sequence, and is one of the first NMR observations of RNA 2'-OH resonances.

Base Composition↗
PubMed1995

Divalent metal ion binding to a conserved wobble pair defining the upstream site of cleavage of group I self-splicing introns.

The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition.

Base Composition↗
PubMed1995

Novel three-dimensional 1H-13C-31P triple resonance experiments for sequential backbone correlations in nucleic acids.

Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H-13C-31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H-31P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihedral angle constraints. By relying on 1H-31P rather than 13C-31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H-31P and 13C-31P couplings provide dihedral angle constraints for RNA structure determination.

Base Sequence↗
PubMed1995

Novel techniques in nuclear magnetic resonance for nucleic acids.

Recent techniques for the efficient preparation of isotopically labelled RNA of desired sequence represent a dramatic step forward for NMR of nucleic acids. Three- and four-dimensional NMR experiments greatly facilitate spectral analysis and quantification of structural constraints. Backbone-driven assignment procedures have been introduced to parallel the powerful assignment methods introduced for work with proteins. Additional structural information to complement interproton distances, namely scalar coupling constants defining the backbone conformation, can be obtained using isotopically labelled oligonucleotides. The additional interproton distance and dihedral angle constraints resolved in higher-dimensional spectra will enable the determination of larger DNA and RNA structures and also increase accuracy and precision.

DNA↗
PubMed1995

Exceptionally stable nucleic acid hairpins.

Hairpins represent the dominant secondary structure element in RNA. Certain sequences are found with exceptional frequency in many RNAs and are characterized by exceptionally high thermodynamic stability. Stable RNA hairpins define nucleation sites for folding, determine tertiary interactions in RNA enzymes, protect mRNAs from degradation, and are recognized by RNA-binding proteins. The structures of several stable DNA and RNA hairpins have revealed networks of stabilizing interactions within the hairpin loop: non-Watson-Crick base pairs and base-phosphate and base-sugar contacts. The unusual stability of these structural elements can be used to stabilize RNA and DNA structures and to protect antisense oligonucleotides and mRNAs against exonucleolytic degradation.

Base Sequence↗
PubMed1994

NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.

The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.

Amino Acid Sequence↗
PubMed1993

The conformation of loop E of eukaryotic 5S ribosomal RNA.

The solution structure of a 27-nucleotide duplex, including the internal loop E from Xenopus laevis 5S ribosomal RNA, has been studied by two-dimensional NMR spectroscopy, followed by restrained molecular dynamics. The highly conserved internal loop closes to form a G.A base pair and a reverse-Hoogsteen A.U base pair. Extensive interstrand stacking between these uncommon base pairs provides a structural explanation for an interstrand ultraviolet-induced cross-link. A guanosine residue is bulged into the major groove and may form a base-triple with the adjacent reverse-Hoogsteen A.U pair. The structure of the less highly-conserved portion of the loop is less well-defined by the NMR data. A single-nucleotide deletion mutant has a very different, open conformation without mismatched base pairs [Varani, G., Wimberly, B., & Tinoco, I. Jr. (1989) Biochemistry 28, 7760-7772]. The implications of the structure for binding of the transcription factor TFIIIA and the cytotoxin alpha-sarcin are discussed.

Animals↗
PubMed1991

Structure of an unusually stable RNA hairpin.

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.

Base Sequence↗
PubMed1990

Solution structure of an unusually stable RNA hairpin, 5'GGAC(UUCG)GUCC.

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.

Base Composition↗
PubMed1989

Conformation and dynamics of an RNA internal loop.

The conformation and the dynamics of an RNA oligonucleotide (26 nucleotides) which is a model for loop E in eukaryotic 5S RNA have been investigated by one- and two-dimensional NMR. The central portion of the oligonucleotide contains two G A oppositions, a common feature of ribosomal RNAs. The exchangeable proton spectrum indicates that an internal loop separates two stems of four and five base pairs. This observation is not consistent with structures for loop E containing mismatched G.A base pairs proposed from chemical and enzymatic studies on Xenopus laevis 5S RNA. The nonexchangeable proton spectrum has been assigned by two-dimensional NMR. Scalar couplings from correlated experiments and interproton distances from NOESY experiments at short mixing times have been used to determine glycosidic angles, sugar puckers, and other conformational features. The conformation of the stems is very close to standard A-form RNA, and extensive base stacking continues into the internal loop. This result provides a structural basis for the large favorable enthalpy of duplex formation determined in thermodynamic studies. Unusual structural and dynamic features are localized in the nucleotides connecting the loop to the stems.

Animals↗
PubMed1988

The TFIIIA recognition fragment d(GGATGGGAG).d(CTCCCATCC) is B-form in solution.

The deoxyoligonucleotide d(GGATGGGAG).d(CTCCCATCC) is a portion of the gene recognition sequence of transcription factor IIIA (TFIIIA). The crystal structure of this oligonucleotide was shown to be A-form (Mc Call, M., Brown, T., Hunter, W.N., and Kennard, O. 1986 Nature 322, 661-664). The present study employs NMR, optical, chemical and enzymatic techniques to investigate the solution structure of this DNA 9-mer. NMR COSY experiments indicate 16 of the 18 residues are predominantly south (C2'-endo) sugar conformation. NMR NOESY indicates glycosidic angles in the range predicted for B-form DNA as opposed to A-form. Related DNA and RNA self-complementary 18-mer sequences, d(GGATGGGAGC-TCCCATCC), with U substituted for T in RNA, were studied by circular dichroism. CD spectra support B-form structures for the DNA 9-mer and the DNA 18-mer, and A-form for the RNA 18-mer. High trifluoroethanol concentrations induce a B- to A-form transition in the DNA oligonucleotides. Enzymatic and chemical probes also illustrate significant differences between the DNA and the RNA oligonucleotides. We find no evidence to support an A-form conformation for the TFIIIA recognition sequence d(GGATGGGAG).d(CTCCCATCC) in solution.

Binding Sites↗
PubMed1987

Nonspecific interactions in dye binding to DNA. Influence of alcohols and amides.

The binding of a few drugs (ethidium bromide, propidium diiodide, proflavine and actinomycin D) to DNA has been investigated in aqueous solutions to which cosolvents of different polarity have been added. It is found that both alcohols (less polar than water) and amides (more polar) lower the binding constant according to a linear relationship between the intercalation free energy and cosolvent concentration. The main action of cosolvents cannot be described in terms of electrostatic effects, since they predict much smaller changes in the binding constant than those observed. It appears instead that relevant solvation effects are responsible for the binding strength of the different dyes to DNA. As a general result, it is found that solvation effects largely contribute to the intercalation free energy, thereby weakening the influence of nonspecific interactions at the intercalation site.

Alcohols↗