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PubMed2005

Calculation of the binding free energy for magnesium-RNA interactions.

The nature of the interaction between nucleic acids and divalent ions in solution is complex. It includes long-range electrostatic and short-range nonelectrostatic forces. Water molecules can be in an inner coordination shell that intervenes between the ion and its binding site. This work describes a method for calculating the binding free energy and applies it to a specific Mg-RNA system in the presence of monovalent salt. The approach combines high-level ab initio theory with Poisson-Boltzmann calculations and provides an accurate description of all terms of the binding free energy for magnesium ions located at the RNA surface (including nonelectrostatic interactions). Some alternative macroscopic approaches are also discussed.

Animals↗

PubMed1999

RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT.

Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria. Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template. We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography. In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members. Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain. Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10(-8) M. The crystal structure of LicT-CAT has been determined at 1.8 A resolution and compared to that of SacY-CAT. Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel beta-sheet that stacks against the beta-sheet of the other monomer in a very conserved manner. Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges. Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures.

Amino Acid Sequence↗

PubMed1996

Two structurally different RNA molecules are bound by the spliceosomal protein U1A using the same recognition strategy.

BACKGROUND: Human U1A protein binds to hairpin II of U1 small nuclear RNA (snRNA) and, together with other proteins, forms the U1 snRNP essential in pre-mRNA splicing. U1A protein also binds to the 3' untranslated region (3'UTR) of its own pre-mRNA, inhibiting polyadenylation of the 3'end and thereby downregulating its own expression. The 3'UTR folds into an evolutionarily conserved secondary structure with two internal loops; one loop contains the sequence AUUGCAC and the other its variant AUUGUAC. The sequence AUUGCAC is also found in hairpin II of U1 snRNA; hence, U1A protein recognizes the same heptanucleotide sequence in two different structural contexts. In order to better understand the control mechanism of the polyadenylation process, we have built a model of the U1A protein-3'UTR complex based on the crystal structure of the U1A protein-hairpin II RNA complex which we determined previously. RESULTS: In the crystal structure of the U1A protein-hairpin II RNA complex the AUUGCAC sequence fits tightly into a groove on the surface of U1A protein. The conservation of the heptanucleotide in the 3'UTR strongly suggests that U1A protein forms identical sequence-specific contacts with the heptanucleotide sequence when complexed with the 3'UTR. The crystal structure of the hairpin II complex and the twofold symmetry in the 3'UTR RNA provide sufficient information to restrict the conformation of the 3'UTR RNA and have enabled us to build a model of the 3'UTR complex. CONCLUSIONS: In the U1A-3'UTR complex, sequence-specific interactions are made entirely by the conserved heptanucleotide and the last base pair (C:G) of the stem. The structure is stabilized by protein-protein contacts and by electrostatic interactions between basic amino acids of the protein and the phosphate backbone of the RNA stem regions. The formation of a protein dimer necessary for the inhibition of poly(A) polymerase requires a conformational change of the C termini of the proteins upon RNA binding. This mechanism could prevent the inhibition of poly(A) polymerase by free U1A protein. The model is consistent with biochemical data, and the protein-protein interactions within the 3'UTR complex account for the cooperativity of U1A protein binding to the 3'UTR. The model also serves as an important structural guide for designing further experiments to understand the interaction between the U1A-3'UTR complex and poly(A) polymerase.

Base Sequence↗

PubMed2005

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1.

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.

Alternative Splicing↗

PubMed2005

Diffusional encounter of barnase and barstar.

We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.

Bacterial Proteins↗

PubMed1977

Ionic regulation in genetic translation systems.

The polyelectrolyte theory can provide an interpretation of the interdependence of pH, ionic strength, and polyamines one observes in the activity of ribonuclease acting on RNA. According to this theory: (i) A nucleic acid-enzyme complex and the suspending medium may be considered as two phases in equilibrium, even though within limits, the complex is soluble in water. (ii) The enzymatic catalysis is under tight control of the electrostatic potential generated by the system. Consequently, modification in electrostatic potential will induce a concomitant change in activity. (iii) The electrostatic potential can be modified through action on the system of "modulators", either "external" (ionic strength, pH, temperature, etc.) or "internal" (specific ligands, substrates, protein factors, etc.). Similarities between the reaction of ribonuclease (ribonuclease 3'-pyrimidino-oligonucleotidohydrolase; EC 3.1.4.22) and RNA and those observed with highly organized systems catalyzing DNA, RNA, and protein synthesis suggest that the electrostatic potential also provides an important regulatory mechanism in genetic translation. In this view, an essential function of nucleic acids is to provide their enzyme partners with polyanionic microenvironments within which their catalytic activities are controlled by variation in physicochemical parameters, including the proton concentration induced through "modulation" of the local electrostatic potential.

Catalysis↗

PubMed2005

Solution structure of the Vts1 SAM domain in the presence of RNA.

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.

Amino Acid Motifs↗

PubMed1983

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects.

Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1 M MgCl2, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacke purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl2 staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl2 were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.

Animals↗

PubMed2002

Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution.

The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes. Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA-DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain. This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130 degrees rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation. A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA-DNA hybrid is completely buried in a newly formed, deep protein cavity. Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions. The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases.

Amino Acid Sequence↗

PubMed2001

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.

Animals↗

PubMed1994

Applications of statistical criteria in protein sequence analysis: case study of yeast RNA polymerase II subunits.

We have recently proposed statistical techniques to identify unusual protein sequence features. Extensive mapping of these features to particular groups of proteins may afford new ways of protein classification. Here we present a case study of such analysis by discussing special features of the amino acid sequences of yeast RNA polymerase II, the first eukaryotic RNA polymerase for which all subunits have been sequenced. Specific new suggestions derived from this analysis include: (i) based on unusual charge configurations in some of the sequences, electrostatic forces may play a significant role in subunit interactions; (ii) RPB4, on account of similar charge distribution, may well be grouped together with RNA polymerase II transcription initiation factors.

Amino Acid Sequence↗

PubMed1984

Molecular model for messenger RNA splicing.

A molecular model is presented for a messenger RNA (mRNA) "splice region." The model requires cation coordination to reduce backbone-backbone electrostatic repulsion and it allows for every base residue on the pre-mRNA to be stacked in A-form helical geometry with a recognition element on the intron or exon (or both) sides of the splice junction. The two nucleotides involved in the initial steps of the cleavage-ligation mechanism must adopt a non-A-form geometry, which ideally positions reactive groups on the pre-mRNA for the necessary catalytic chemistry. The model is also consistent with available biochemical data on splicing reactions.

Adenosine Triphosphate↗

PubMedDate not supplied

Functional aspects of ribosomal proteins.

A short personal recollection of HG Wittmann is given with emphasis on his basic contribution to the structure of the ribosome, in particular the ribosomal proteins. With these considerations in mind, two interrelated problems are reviewed here. The first relates to the internal symmetry both in tRNA and in the tetrameric L12-protein complex. The second problem to be addressed relates to the dynamics of transfer RNA in the ribosome and the role of L12 proteins in this process. The importance of electrostatic repulsion in the maintenance of the mutual spatial orientation of tRNAs and L12 in the ribosome is emphasized in relation to a pendulum model for how L12 may steer translocation.

Amino Acid Sequence↗

PubMed1989

Visualization of protein-nucleic acid interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9 A resolution by X-ray fiber diffraction.

The structure of tobacco mosaic virus (TMV) has been determined by fiber diffraction methods at 2.9 A resolution, and refined by restrained least-squares to an R-factor of 0.096. Protein-nucleic acid interactions are clearly visible. The final model contains all of the non-hydrogen atoms of the RNA and the protein, 71 water molecules, and two calcium-binding sites. Viral disassembly is driven by electrostatic repulsions between the charges in two carboxyl-carboxylate pairs and a phosphate-carboxylate pair. The phosphate-carboxylate pair and at least one of the carboxyl-carboxylate pairs appear to be calcium-binding sites. Nucleotide specificity, enabling TMV to recognize its own RNA by a repeating pattern of guanine residues, is provided by two guanine-specific hydrogen bonds in one of the three base-binding sites.

Amino Acids↗

PubMed2004

Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode.

The influenza virus nonstructural protein 1 encoded by influenza A virus (NS1A protein) is a multifunctional protein involved in both protein-protein and protein-RNA interactions. NS1A binds nonspecifically to double-stranded RNA (dsRNA) and to specific protein targets, and regulates several post-transcriptional processes. The N-terminal structural domain corresponding to the first 73 amino acids of the NS1 protein from influenza A/Udorn/72 virus [NS1A(1-73)] possesses all of the dsRNA binding activities of the full-length protein. Both NMR and X-ray crystallography of this domain have demonstrated that it is a symmetric homodimer which forms a six-helix chain fold, a unique structure that differs from that of the predominant class of dsRNA-binding domains, termed dsRBDs, that are found in a large number of eukaryotic and prokaryotic proteins. Here we describe biophysical experiments on complexes containing NS1A(1-73) and a short 16 bp synthetic dsRNA duplex. From sedimentation equilibrium measurements, we determined that the dimeric NS1A(1-73) binds to the dsRNA duplex with a 1:1 stoichiometry, yielding a complex with an apparent dissociation constant (K(d)) of approximately 1 microM. Circular dichroism and nuclear magnetic resonance (NMR) data demonstrate that the conformations of both NS1A(1-73) and dsRNA in the complex are similar to their free forms, indicating little or no structural change in the protein or RNA upon complex formation. NMR chemical shift perturbation experiments show that the dsRNA-binding epitope of NS1A(1-73) is associated with helices 2 and 2'. Analytical gel filtration and gel shift studies of the interaction between NS1A(1-73) and different double-stranded nucleic acids indicate that NS1A(1-73) recognizes canonical A-form dsRNA, but does not bind to dsDNA or dsRNA-DNA hybrids, which feature B-type or A/B-type intermediate conformations, respectively. On the basis of these results, we propose a three-dimensional model of the complex in which NS1A(1-73) sits astride the minor groove of A-form RNA with a few amino acids in the helix 2-helix 2' face forming an electrostatically stabilized interaction with the phosphodiester backbone. This mode of dsRNA binding differs from that observed for any other dsRNA-binding protein.

Carbon Isotopes↗

PubMed1988

Two-dimensional crystals of Escherichia coli RNA polymerase holoenzyme on positively charged lipid layers.

Escherichia coli RNA polymerase holoenzyme forms two-dimensional crystals when adsorbed to positively charged lipid layers at the air/water interface. Adsorption of the protein is driven by electrostatic interactions between the positively charged lipid surface and the polymerase molecule, which has a net negative charge. Crystallization is dependent on the adsorption and concentration of RNA polymerase on fluid lipid surfaces. Image analysis of electron micrographs of crystals in negative stain, which diffract to 30 A resolution, shows irregularly shaped protein densities about 100 x 160 A, consistent with the dimensions of single polymerase molecules.

DNA-Directed RNA Polymerases↗

PubMed1995

R-loop stability as a function of RNA structure and size.

The sequence-specific formation of R-loops can be assayed using RNAs which overlap a HindIII cleavage site in a 3.5 kb plasmid. Chemical modification of the displaced DNA strand has permitted stabilization of these R-loops and allowed a systematic investigation of the dependence of these triple-stranded structures on the chain length and structure of the input RNA. RNAs as short as 50 nt form stable R-loops if 5-allylamine uridines (Uaa-RNA) are used in place of normal uridines; normal RNAs must be 100 nt long to form R-loops quantitatively. Since acetic anhydride decreases the hybridization efficiency of Uaa-RNAs, the positive charge of the RNAs must diminish the electrostatic repulsion of the three negatively charged phosphodiester backbones. The dependence of R-loop stability on the length of RNA can be stimulated with a random walk model, which also applies to strand migration within Holiday junctions. R-loop hybridization provides a versatile method to generate single-stranded DNA in a sequence-selective manner.

DNA↗

PubMed2006

Comparison of properties of particles of Cucumber mosaic virus and Tomato aspermy virus based on the analysis of molecular surfaces of capsids.

The plant RNA viruses Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) (genus Cucumovirus) have similar icosahedral particles, the crystal structures of which have been reported recently. Similarity in particle structure agrees with reports of stable capsids assembled from their capsid proteins and of viable recombinant viruses with chimeric capsid proteins derived from CMV and TAV. However, differences between the cucumoviruses have been reported for physicochemical properties. Here, structural and electrostatic features of the molecular surfaces are studied to investigate their relationship with these observations. Two coat-protein recombinants with structures modelled by taking CMV and TAV as templates were also included in the analysis. Results show that there exists an external region of negative electrostatic potential that has arisen from strictly conserved charged residues situated near the external HI loop of the subunits in the capsomers. This negative domain surrounds the fivefold and quasi-sixfold axes and locates above regions of positive potential that extend to cover, nearly homogeneously, the inner surface of capsids, where interaction with encapsidated RNA occurs. Differences between the outer electrostatic distributions in CMV and TAV explain the distinct response of both viruses to variations in physicochemical conditions required for particle stability and are essential to rationalize the biological activity of the coat-protein recombinants, in spite of their seemingly distinct electrostatic characteristics.

Capsid↗