A role for aromatic amino acids in the binding of Xenopus ribosomal protein L5 to 5S rRNA.

The formation of the Xenopus L5-5S rRNA complex depends on nonelectrostatic interactions. Fluorescence assays with 1-anilino-8-naphthalenesulfonate demonstrate that a hydrophobic region on L5 becomes exposed upon removal of bound 5S rRNA by treatment with ribonucleases. Several conserved aromatic amino acids, mostly tyrosines, were identified by comparative sequence analysis and changed individually to alanine. Substitution with alanine at any of three positions, Y86, Y99, or Y226, essentially abolishes RNA-binding activity, whereas those made at Y95 and Y207 have more modest effects. Replacement with phenylalanine at Y86 and Y226 does not change binding affinity, indicating that the aromatic ring of the side chain, not the hydroxyl group, is the critical functionality for binding. Alternatively, the phenolic hydroxyls at Y99 and Y207 do contribute to binding. The structural integrity of the mutant proteins was assessed using thermal denaturation and limited digestion with proteases. The T(m) of Y99A is 10 degrees C lower than that of the wild-type protein, and there are some differences in the protease digestion patterns that together indicate the structure of this mutant has been significantly perturbed. The structures of the other variants are not detectably different from the wild-type protein. These results provide evidence that intermolecular stacking interactions involving at least two tyrosine residues, Y86 and Y226, are necessary for formation of the L5-5S rRNA complex and can account, at least in part, for the contribution nonelectrostatic interactions make to the free energy of binding.

The structure of helix III in Xenopus oocyte 5 S rRNA: an RNA stem containing a two-nucleotide bulge.

The solution structure of an oligonucleotide containing the helix III sequence from Xenopus oocyte 5 S rRNA has been determined by NMR spectroscopy. Helix III includes two unpaired adenosine residues, flanked on either side by G:C base-pairs, that are required for binding of ribosomal protein L5. The consensus conformation of helix III in the context provided by this oligonucleotide has the two adenosine residues located in the minor groove and stacked upon the 3' flanking guanosine residue, consistent with biochemical studies of free 5 S rRNA in solution. A distinct break in stacking that occurs between the first adenosine residue of the bulge and the flanking 5' guanosine residue exposes the base of the adenosine residue in the minor groove and the base of the guanosine residue in the major groove. The major groove of the helix is widened at the site of the unpaired nucleotides and the helix is substantially bent; nonetheless, the G:C base-pairs flanking the bulge are intact. The data indicate that there may be conformational heterogeneity centered in the bulge region. The corresponding adenosine residues in the Haloarcula marismortui 50 S ribosomal subunit form a dinucleotide platform, which is quite different from the motif seen in solution. Thus, the conformation of helix III probably changes when 5 S rRNA is incorporated into the ribosome.

A proline-rich protein binds to the localization element of Xenopus Vg1 mRNA and to ligands involved in actin polymerization.

A 340 nucleotide element within the 3' untranslated region of Vg1 mRNA determines its localization to the vegetal cortex of Xenopus oocytes. To identify protein factors that bind to this region, we screened a cDNA expression library with an RNA probe containing this sequence. Five independent isolates encoded a protein (designated Prrp for proline-rich RNA binding protein) having two RNP domains followed by multiple polyproline segments. Prrp and Vg1 mRNAs are co-localized to the vegetal cortex of stage IV oocytes, substantiating an interaction between the two in vivo. Prrp also associates with VegT mRNA, which like Vg1 mRNA uses the late localization pathway, but not with Xcat-2 or Xwnt-11 mRNAs, which use the early pathway. The proline-rich domain of Prrp interacts with profilin, a protein that promotes actin polymerization. Prrp can also associate with the EVH1 domain of Mena, another microfilament-associated protein. Since the anchoring of Vg1 mRNA to the vegetal cortex is actin dependent, one function of Prrp may be to facilitate local actin polymerization, representing a novel function for an RNA binding protein.

Inhibition of RNA polymerase III transcription by a ribosome-associated kinase activity.

Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.

Binding of neomycin to the TAR element of HIV-1 RNA induces dissociation of Tat protein by an allosteric mechanism.

Neomycin inhibits the binding of Tat-derived peptides to the trans-activating region (TAR) of HIV-1 RNA. Kinetic studies reveal that neomycin acts as a noncompetitive inhibitor that can bind to the Tat-TAR complex and increase the rate constant (koff) for dissociation of the peptide from the RNA. Neomycin effects a conformational change in the structure of TAR that can be detected by circular dichroism spectroscopy. The increase in ellipticity measured at 265 nm upon binding of the aminoglycoside is opposite to the decrease seen when Tat peptides bind to the RNA. Thus, the structural transition induced by neomycin is apparently incompatible with the binding of Tat and underlies the inhibitory action of the antibiotic. The binding site for neomycin on TAR was identified in ribonuclease protection experiments and is located in the stem immediately below the three-nucleotide bulge that serves as the primary identity element for Tat. Apparent protection of residues in the bulge by neomycin may represent additional contacts to the aminoglycoside, but more likely result from changes in the structure of this region when the ligand binds to the RNA. Binding assays using variants of TAR in which inosine residues were substituted for guanosine residues support the results from the ribonuclease protection experiments. Inosine substitutions in the lower stem, but not the upper stem, decrease the binding constant for neomycin by approximately 100-fold. Neither of these variants affected the binding affinity of Tat peptide. In addition, these latter experiments suggest that the aminoglycoside may be located in the minor groove of the stem. This mode of association may be a critical aspect of neomycin's ability to bind to the Tat-TAR complex and could serve as a guide for the design of other drugs that bind to specific RNA targets as noncompetitive inhibitors.

Analysis of the binding of Xenopus transcription factor IIIA to oocyte 5 S rRNA and to the 5 S rRNA gene.

Binding of transcription factor IIIA (TFIIIA) to site-specific mutants of Xenopus oocyte 5 S rRNA has been used to identify important recognition elements in the molecule. The putative base triple G75:U76:A100 appears to determine the conformation of the loop E region whose integrity is especially important for binding of the factor. Proximal substitutions in helices IV and V indicate that the proper folding of loop E is also dependent on these structures. Mutations in helix V affect binding of TFIIIA to 5 S rRNA and to the gene similarly and provide evidence that zinc finger 5 makes sequence-specific contact through the major groove of both nucleic acids. Although fingers 1-3 are positioned along helix IV and loop D, mutations in this region, including those that disrupt the tetraloop or close the opening in the major groove of the helix created by the U80:U96 mismatch, have no impact on binding. Substitutions made at stem-loop junctions in the arm of the RNA comprised of helix II-loop B-helix III display minor decreases in affinity for TFIIIA. Despite the alignment of the factor along nearly the entire length of 5 S rRNA, the essential elements for high affinity binding are limited to the central region of the molecule. Analysis of the corresponding mutations in the gene confirm that box C and the intermediate element provide the high affinity sites for binding of the factor to the DNA. Despite the small thermodynamic contribution made by contacts to box A, mutations made in this element can cause substantial changes in the orientation of the carboxyl-terminal fingers along the 5'-end of the internal control region.

Analysis of the binding of Xenopus ribosomal protein L5 to oocyte 5 S rRNA. The major determinants of recognition are located in helix III-loop C.

Xenopus ribosomal protein L5 was expressed in Escherichia coli and exhibits high affinity (Kd = 2 nM) and specificity for oocyte 5 S rRNA. The pH dependence of the association constant for the complex reveals an ionization with a pK alpha value of 10.1, indicating that tyrosine and/or lysine residues are important for specific binding of L5 to the RNA. Formation of the L5.5 S rRNA complex is remarkably insensitive to ionic strength, providing evidence that nonelectrostatic interactions make significant contributions to binding. Together, these results suggest that one or more tyrosine residues may form critical contacts through stacking interactions with bases in the RNA. In order to locate recognition elements within 5 S rRNA, we measured binding of L5 to a collection of site-specific mutants. Mutations in the RNA that affected the interaction are confined to the hairpin structure comprised of helix III and loop C. Earlier experiments with a rhodium structural probe had shown that the two-nucleotide bulge in helix III and the intrinsic structure of loop C create sites in the major groove that are opened and accessible to stacking interactions with the metal complex. In the present studies, we detect a correlation between the intercalative binding of the rhodium complex to mutants in the hairpin and binding of L5, supporting the proposal that binding of the protein is mediated, in some part, by stacking interactions. Furthermore, the results from mutagenesis establish that, despite overlapping binding sites on 5 S rRNA, L5 and transcription factor IIIA utilize distinct structural elements for recognition.

Chemical nucleases: their use in studying RNA structure and RNA-protein interactions.

Metal complexes that cleave nucleic acids provide a new means to study RNA structure and RNA-protein interactions. Methods that use these chemical nucleases help compensate for the limitations of other techniques used to determine structure. Because the ligands that coordinate the metal generally control the cleavage selectivity of these complexes, it has become possible to design nucleolytic reagents that target specific higher-order structures. In combination with site-directed mutagenesis these conformation-specific probes can be used to delineate long-range interactions. Alternatively, complexes that cut irrespective of sequence and secondary structure have been used in protection (foot-printing) experiments to locate protein binding sites. Because each position of the nucleic acid is susceptible to cleavage, the protection pattern yields a highly resolved definition of the contact site between the protein and RNA. In other applications, metal complexes have been conjugated to functional moieties such as oligonucleotides, peptides, or substrate analogs to direct their binding to a distinct site on a specific RNA molecule. This latter strategy holds significant therapeutic promise for the destruction of pathogenic RNAs.

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural polymorphism in the major groove of a 5S RNA gene complements the zinc finger domains of transcription factor IIIA.

Metal complexes that bind to DNA on the basis of shape-selection have been used to map the conformational features of the DNA binding site for transcription factor IIIA. Conformationally distinct segments are detected on the 5S rRNA gene that correspond closely to the binding sites identified for the individual zinc finger domains of the protein. The local conformations are characterized by a major groove opened because of a change in base pair inclination and/or displacement at a central 5'-pyrimidine-purine-3' step, flanked by a widened minor groove, as would arise at the junctions between alternating B- and A-like DNA segments. Docking experiments with a consensus structure of a zinc finger reveal that the mixed A-B binding site accommodates the peptide domain better than either canonical B- or A-DNA helices. The close structural matching of the conformational variations in the 5S rDNA both to the proposed sites of zinc finger binding and to the shape of an individual zinc finger domain points to DNA structural polymorphism as providing an important determinant in recognition. In particular, shape selection in the 5' half of the internal control region may orient the multiple finger domains.

The use of chemical nucleases to analyze RNA-protein interactions. The TFIIIA-5 S rRNA complex.

The complex of Xenopus transcription factor IIIA (TFIIIA) with 5 S rRNA was analyzed in nuclease protection experiments using hydroxyl radical. The protection pattern reveals that TFIIIA interacts with a substantial amount of the RNA molecule, with close association between the factor and the arm of the RNA composed of helix IV-loop E-helix V. Additional sites of protection punctuate the other arm of the molecule. Important contact sites within the complex were identified in "missing nucleoside" experiments. Random single-nucleoside gaps were introduced into 5 S rRNA using either Fe[EDTA]2-/H2O2 or bis(1,10-phenanthroline)copper(I). This modified RNA was allowed to bind to TFIIIA in an exchange reaction, and, afterward, bound and unbound fractions were separated on nondenaturing polyacrylamide gels. Missing nucleoside positions specifically enriched in the unbound fraction of RNA are located in the two strands that comprise loop E. These are not necessarily sites of sequence-specific contacts, but rather may constitute a region of secondary structure essential to recognition and binding of TFIIIA to 5 S rRNA.

Conformational studies of the nucleic acid binding sites for Xenopus transcription factor IIIA.

The CD spectrum of a restriction fragment that contains a single copy of a Xenopus borealis somatic 5 S rRNA gene, like those of two smaller fragments from the binding site for transcription factor IIIA (TFIIIA), is that of B-form DNA. Under dehydrating conditions (76% ethanol) the 5 S rDNA undergoes a transition into an A conformation. The spectra of the three fragments, however, do exhibit some perturbation in that the longwave positive peaks are shifted to shorter wavelengths and have enhanced rotational strength compared with reference B-form spectra. The helical repeats measured for the smaller fragments indicate that the helix winding angle can account for these features in the CD spectra. We suggest that G:C-rich boxes that punctuate not only the TFIIIA binding site but the whole 5 S gene are responsible for the conformational perturbation manifest in the CD spectra and may play a role in the recognition of the DNA by the factor. The spectrum of the gene is unchanged in the presence of TFIIIA, indicating that the structural heterogeneity of the DNA persists upon complex formation. The CD spectra of native TFIIIA.5 S rRNA particles isolated from oocytes and of particles reconstituted in vitro are identical and only moderately different from the spectrum of free 5 S rRNA, suggesting that the protein effects only limited changes in the secondary and/or tertiary structure of the RNA. The helical structure of the two binding sites is discussed with respect to a common mode of interaction of TFIIIA with DNA and RNA.

Little risks and big fears.

Various aspects of regulatory decisions involving the concepts of "unacceptably hazardous" and "acceptably safe" as related to the growing emphasis on the legal terminology of de minimis are outlined. It is concluded that with rare exceptions a proper public policy for risk evaluations is far from implementation.

Use of the cytotoxic nuclease alpha-sarcin to identify the binding site on eukaryotic 5 S ribosomal ribonucleic acid for the ribosomal protein L5.

Treatment of the large subunit of eukaryotic ribosomes with EDTA releases a 7 S ribonucleoprotein complex that contains protein L5 and 5 S rRNA. The region of rat 5 S rRNA that is protected by protein L5 from digestion with the ribonuclease alpha-sarcin includes nucleotides 1-10, 12-22, and 64-116. This domain approximates the combined binding sites for the three proteins (L5, L18, and L25) that associate with Escherichia coli 5 S rRNA. In addition, the region of 5 S rRNA protected by rat ribosomal protein L5 corresponds closely to that occupied by Xenopus transcription factor IIIA.

Identification of the binding site on 5S rRNA for the transcription factor IIIA: proposed structure of a common binding site on 5S rRNA and on the gene.

Transcription factor IIIA interacts specifically with an internal control region of Xenopus 5S ribosomal RNA genes and is also a component, along with 5S rRNA, of a 7S ribonucleoprotein particle present in previtellogenic oocytes. We have determined the region of the 5S rRNA in the 7S ribonucleoprotein complex that is protected by the transcription factor from digestion with the ribonuclease alpha-sarcin. The binding site for factor IIIA extends from nucleotide 64 through nucleotide 116; the protected region includes two CCUGG helices separated by 11 nucleotides. The same helices occur in the factor IIIA binding site in the 5S rRNA gene and may constitute a common structural feature recognized by the protein in the gene and in the gene product.

Kinetic studies of the reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate.

The reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate (NHxDPH) has been examined by stopped-flow kinetic methods. Like reduced glutathione or NADPH, this pyridine nucleotide generates the catalytically active two-electron reduced form of the enzyme. This reductive half-reaction with NHxDPH has only one detectable kinetic step which shows saturation kinetics (Kd = 76 microM), and has a limiting rate constant of 56 s-1. Comparison of stopped-flow and steady-state turnover data indicates that the reductive half-reaction is rate-limiting in the overall catalytic reaction. No evidence was found for a preequilibrium charge-transfer complex between NHxDPH and the active site FAD, like that seen when NADPH is the electron donor.