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Biomedical subjects

G Varani

Publications and source records attributed to G Varani.

At least 19 recordsLinked to original sources
PubMed1999

Structure-based and combinatorial search for new RNA-binding drugs.

The discovery by structure-based and combinatorial methods of new RNA-binding drugs presents great opportunities for pharmacological development against drug-resistant bacterial and viral pathogens. A handful of recent RNA structures and more numerous studies of the interaction of combinatorial libraries and oligomeric RNA-binding compounds are providing the foundation for effective RNA-targeted drug discovery programs.

Chemistry, Pharmaceutical↗
PubMed1998

Determination of the NMR structure of the complex between U1A protein and its RNA polyadenylation inhibition element.

RNA-protein recognition is critical to post-transcriptional regulation of gene expression, yet poorly understood at the molecular level. The relatively slow progress in understanding this important area of molecular biology is due to difficulties in obtaining good-quality crystals and derivatives, and in preparing samples suitable for NMR investigation. The determination of the structure of the complex between the human U1A protein and its polyadenylation inhibition element is described here. In this paper, we describe the sample preparation, spectral assignments, construction of the NOE-based distance constraints and methodology for calculating the structure of the complex. The structure was determined to an overall precision of 2.03 A (for all ordered regions), and 1.08 A for the protein-RNA interface. The patterns of hydrogen bonding and hydrophobic interactions at the interface were analysed statistically using the final ensemble of 31 structures.

Amino Acid Sequence↗
PubMed1998

RNA recognition by RNP proteins during RNA processing.

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein folds. Proteins containing RNP domains function in important steps of posttranscriptional regulation of gene expression by directing the assembly of multiprotein complexes on primary transcripts, mature mRNAs, and stable ribonucleoprotein components of the RNA processing machinery. The diverse functions performed by these proteins depend on their dual ability to recognize RNA and to interact with other proteins, often utilizing specialized auxiliary domains. Crystallographic and NMR structures of several RNP domains and a handful of structures of RNA-protein complexes have begun to reveal the molecular basis for RNP-RNA recognition.

Alternative Splicing↗
PubMed1997

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.

Amino Acid Sequence↗
PubMed1997

A cap for all occasions.

The 5' end of each polymerase II transcript is capped by a methylated guanosine triphosphate. The cap earmarks the mRNA for subsequent processing and nucleocytoplasmic transport, protects the mRNA from degradation and promotes efficient initiation of protein synthesis. The recently solved structures of capping enzymes and cap-protein complexes shed light on how the 5' ends of mRNAs are modified, and reveals the mechanisms by which the cap is recognized and how it functions in a diverse range of processes.

Models, Biological↗
PubMed1997

Structure of the acceptor stem of Escherichia coli tRNA Ala: role of the G3.U70 base pair in synthetase recognition.

The fidelity of translation of the genetic code depends on accurate tRNA aminoacylation by cognate aminoacyl-tRNA synthetases. Thus, each tRNA has specificity not only for codon recognition, but also for amino acid identity; this aminoacylation specificity is referred to as tRNA identity. The primary determinant of the acceptor identity of Escherichia coli tRNAAlais a wobble G3.U70 pair within the acceptor stem. Despite extensive biochemical and genetic data, the mechanism by which the G3.U70 pair marks the acceptor end of tRNAAla for aminoacylation with alanine has not been clarified at the molecular level. The solution structure of a microhelix derived from the tRNAAla acceptor end has been determined at high precision using a very extensive set of experimental constraints (approximately 32 per nt) obtained by heteronuclear multidimensional NMR methods. The tRNAAla acceptor end is overall similar to A-form RNA, but important differences are observed. The G3.U70 wobble pair distorts the conformation of the phosphodiester backbone and presents the functional groups of U70 in an unusual spatial location. The discriminator base A73 has extensive stacking overlap with G1 within the G1.C72 base pair at the end of the double helical stem and the -CCA end is significantly less ordered than the rest of the molecule.

Alanine-tRNA Ligase↗
PubMed1997

An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication.

One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 x 10(6) individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10-30 microM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent transactivation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo.

Anti-HIV Agents↗
PubMed1997

How accurately and precisely can RNA structure be determined by NMR?

The great diversity of RNA biological functions has led to widespread interest in RNA structure. Advances in synthetic and spectroscopic techniques have recently allowed the extension of NMR methods of structure determination to RNA structures of significant size and increased biological significance. However, it has not yet been established how accurately and precisely RNA structure can be determined by NMR. The extensive simulations presented here establish credible limits on accuracy and precision of NMR-derived RNA structures and provide quantitative calibrations to evaluate new structures. Synthetic sets of NMR constraints were generated from a crystallographically-derived ribozyme structure. The target structure was then redetermined using approximations and computational protocols derived from our experimental work. The results demonstrate that the structure of RNA molecules of at least 15,000 Da can be determined with precision and accuracy of 1 to 1.5 A, comparable to values obtained for proteins of similar size. Most encouragingly, it is shown that larger, globular and biologically more important RNA structures can be determined with significantly better accuracy and precision than smaller, elongated structures investigated until now.

Base Composition↗
PubMed1997

Recent solution structures of RNA and its complexes with drugs, peptides and proteins.

The past two years have seen remarkable progress in the study of RNA structure: the predicted era of RNA structural biology has arrived. Crystallographic structures of the hammerhead ribozyme and of a large subunit of a group I self-splicing intron have begun to reveal the structural basis of RNA enzymatic activity. A remarkable number of structures of small RNAs and of complexes with drugs, peptides and one protein domain have been determined by NMR.

Forecasting↗
PubMed1997

Nucleic acids structure and recognition.

A growing understanding of the diversity of structures and biological functions of DNA and RNA, coupled with progress in synthetic and spectroscopic techniques, have allowed remarkable progress in NMR studies of nucleic acids. Here we review novel principles of nucleic acid structure and recognition revealed by these studies and highlight opportunities for future progress.

DNA↗
PubMed1996

Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.

Efficient transcription from the human immunodeficiency virus (HIV) promoter depends on binding of the viral regulatory protein Tat to a cis-acting RNA regulatory element, TAR. Tat binds at a trinucleotide bulge located near the apex of the TAR stem-loop structure. An essential feature of Tat-TAR interaction is that the protein induces a conformational change in TAR that repositions the functional groups on the bases and the phosphate backbone that are critical for specific intermolecular recognition of TAR RNA. We have previously determined a high resolution structure for the bound form of TAR RNA using heteronuclear NMR. Here, we describe a high resolution structure of the free TAR RNA based on 871 experimentally determined restraints. In the free TAR RNA, bulged residues U23 and C24 are stacked within the helix, while U25 is looped out. This creates a major distortion of the phosphate backbone between C24 and G26. In contrast, in the bound TAR RNA, each of the three residues from the bulge are looped out of the helix and U23 is drawn into proximity with G26 through contacts with an arginine residue that is inserted between the two bases. Thus, TAR RNA undergoes a transition from a structure with an open and accessible major groove to a much more tightly packed structure that is folded around basic side chains emanating from the Tat protein.

Antiviral Agents↗
PubMed1996

Specificity of ribonucleoprotein interaction determined by RNA folding during complex formulation.

Many proteins involved in pre-mRNA processing contain one or more copies of a 70-90-amino-acid alphabeta module called the ribonucleoprotein domain. RNA maturation depends on the specific recognition by ribonucleoproteins of RNA elements within pre-mRNAs and small nuclear RNAs. The human U1A protein binds an RNA hairpin during splicing, and regulates its own expression by binding an internal loop in the 3'-untranslated region of its pre-mRNA, preventing polyadenylation. Here we report the nuclear magnetic resonance structure of the complex between the regulatory element of the U1A 3'-untranslated region (UTR) and the U1A protein RNA-binding domain. Specific intermolecular recognition requires the interaction of the variable loops of the ribonucleoprotein domain with the well-structured helical regions of the RNA. Formation of the complex then orders the flexible RNA single-stranded loop against the protein beta-sheet surface, and reorganizes the carboxy-terminal region of the protein to maximize surface complementarity and functional group recognition.

Amino Acid Sequence↗
PubMed1996

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

Amino Acid Sequence↗
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↗