Additional intragenic promoter elements of the Xenopus 5S RNA genes upstream from the TFIIIA-binding site.

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.

Structural requirements of 5S rRNA for nuclear transport, 7S ribonucleoprotein particle assembly, and 60S ribosomal subunit assembly in Xenopus oocytes.

Structural requirements of 5S rRNA for nuclear transport and RNA-protein interactions have been studied by analyzing the behavior of oocyte-type 5S rRNA and of 31 different in vitro-generated mutant transcripts after microinjection into the cytoplasm of Xenopus oocytes. Experiments reveal that the sequence and secondary and/or tertiary structure requirements of 5S rRNA for nuclear transport, storage in the cytoplasm as 7S ribonucleoprotein particles, and assembly into 60S ribosomal subunits are complex and nonidentical. Elements of loops A, C, and E, helices II and V, and bulged and hinge nucleotides in the central domain of 5S rRNA carry the essential information for these functional activities. Assembly of microinjected 5S rRNA into 60S ribosomal subunits was shown to occur in the nucleus; thus, the first requirement for subunit assembly is nuclear targeting. The inhibitory effects of ATP depletion, wheat germ agglutinin, and chilling on the nuclear import of 5S rRNA indicate that it crosses the nuclear envelope through the nuclear pore complex by a pathway similar to that used by karyophilic proteins.

Characterization of the 5 S RNA binding activity of Xenopus zinc finger protein p43.

One major component of the Xenopus 42 S ribonucleoprotein (RNP) storage particle is the p43 protein. The 5 S RNA binding protein is structurally similar to TFIIIA, containing nine zinc finger domains. The RNA binding properties of recombinant p43 were characterized using a nitrocellulose filter binding assay. The experimental conditions necessary for in vitro p43-5 S RNA complex formation include: pH 7.5, 0.1 M KCl and incubation at 22 degrees C. Under these conditions, the protein binds to Xenopus oocyte 5 S RNA with an apparent association constant of 1.61(+/- 0.12) x 10(9) M-1. A series of mutations in 5 S RNA were used to determine which sequence and structural features of the 5 S RNA are required for high affinity binding of p43. The primary contact points for p43 include the sequences and structures of stems II, V and loop D of the 5 S RNA. Although p43 and TFIIIA are structurally similar and are both relatively insensitive to mutations in the 5 S RNA, they do require different features of the 5 S RNA molecule for high affinity binding.

Involvement of "hinge" nucleotides of Xenopus laevis 5 S rRNA in the RNA structural organization and in the binding of transcription factor TFIIIA.

Nucleotides in the bifurcation region of the 5 S rRNA, the junction of the three helical domains, play a central role in determining the coaxial stacking interactions and tertiary structure of the RNA. We have used site-directed mutagenesis of Xenopus laevis oocyte 5 S rRNA to make all possible nucleotide substitutions at three positions in loop A (10, 11 and 13) and at the G66.U109 base-pair at the beginning of helix V. Certain double point mutations were constructed to ascertain the relationship between loop A nucleotides and the G.U base-pair. The importance of the size of the bifurcation region was tested by the creation of a single nucleotide deletion mutant and two single nucleotide insertion mutants. The effects of these mutations on the structure and function of the 5 S rRNA were determined by solution structure probing of approximately half of the mutants with chemical reagents, and by measuring the relative binding affinity of each mutant for transcription factor TFIIIA. Proposed structural rearrangements in the bifurcation region were tested by using a graphic modeling method combining stereochemical constraints and chemical reactivity data. From this work, several insights were obtained into the general problem of helix stacking and RNA folding at complex bifurcation regions. None of the mutations caused an alteration of the coaxial stacking of helix V on helix II proposed for the wild-type 5 S rRNA. However, the formation of a Watson-Crick pair between nucleotide 13 of loop A and nucleotide 66 at the top of helix V does cause a destabilization of the proximal part of this helix. Also, nucleotide 109 at the top of helix V will preferentially pair with nucleotide 10 of loop A rather than nucleotide 66 when both possibilities are provided, without affecting the stability of helix V, even though the G.U pair is disrupted. The effects of these mutations on TFIIIA binding indicate that the bifurcation region is critical for protein recognition. One important feature of the relationship between 5 S rRNA structure and TFIIIA recognition resulting from this study was the observation that any mutation that constrains the bifurcation loop results in a reduced affinity of the RNA for TFIIIA, unless it is compensated for by an increased flexibility elsewhere.

Effect of mutations in domain 2 on the structural organization of oocyte 5 S rRNA from Xenopus laevis.

In order to test and refine the molecular model of Xenopus laevis 5 S rRNA proposed in a previous work, we have synthesized, by site-directed mutagenesis and in vitro transcription, four mutants in the internal loop B and in the hairpin loop C of domain 2. The conformations of these mutant 5 S rRNAs have been tested using a variety of enzymatic and chemical structure-specific probes and computer modeling. The mutations induce conformational changes restricted to the mutated regions. Our results demonstrate unambiguously that the three helical domains of the Y-shaped structure are independent and that loop C possesses an intrinsic conformation, which is not involved in any tertiary long-range interaction. They point to the crucial role of invariant nucleotides in maintaining the intrinsic conformation of the loop and to the effect of sequence on the stability of loop regions.

Computer modeling from solution data of spinach chloroplast and of Xenopus laevis somatic and oocyte 5 S rRNAs.

Detailed atomic models of a eubacterial 5 S rRNA (spinach chloroplast 5 S rRNA) and of a eukaryotic 5 S rRNA (somatic and oocyte 5 S rRNA from Xenopus laevis) were built using computer graphic. Both models integrate stereochemical constraints and experimental data on the accessibility of bases and phosphates towards several structure-specific probes. The base sequence was first inserted on to three-dimensional structural fragments picked up in a specially devised databank. The fragments were modified and assembled interactively on an Evans & Sutherland PS330. Modeling was finalized by stereochemical and energy refinement. In spite of some uncertainty in the relative spatial orientation of the substructures, the broad features of the models can be generalized and several conclusions can be reached: (1) both models adopt a distorted Y-shape structure, with helices B and D not far from colinearity; (2) no tertiary interactions exist between loop c and region d or loop e; (3) the internal loops, in particular region d, contain several non-canonical base-pairs of A.A, U.U and A.G types; (4) invariant residues appear to be more important for protein or RNA binding than for maintaining the tertiary structure. The models are corroborated by footprinting experiments with ribosomal proteins and by the analysis of various mutants. Such models help to clarify the structure-function relationship of 5 S rRNA and are useful for designing site-directed mutagenesis experiments.

Interaction of the RNA binding fingers of Xenopus transcription factor IIIA with specific regions of 5 S ribosomal RNA.

Zinc fingers 4 to 7 of Xenopus transcription factor IIIA (TFIIIA) represent the minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for their effects on the binding affinity of full-length TFIIIA and a polypeptide consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease footprinting was used to compare the binding sites of TFIIIA and zf4-7 on 5 S RNA. The consistency between the data obtained from these two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA bind to a central core region on the 5 S RNA molecule consisting of loop B/helix II/loop A/helix V/region E. This information was used to design a truncated 75-nucleotide-long RNA molecule that retains high affinity for zf4-7. Therefore, we conclude that the specific interaction of TFIIIA with 5 S RNA can be represented by a complex formed between a four zinc finger polypeptide and a truncated 5 S RNA molecule.

Structural studies on site-directed mutants of domain 3 of Xenopus laevis oocyte 5 S ribosomal RNA.

Base substitutions have been introduced into the highly conserved sequences of loops D and E within domain 3 of Xenopus laevis oocyte 5 S rRNA. The effects of these mutations on the solution structure of this 5 S rRNA have been studied by means of probing with nucleases, and with chemical reagents under native and semi-denaturing conditions. The data obtained with these mutants support the graphic model of Xenopus oocyte 5 S rRNA proposed by Westhof et al. In particular, our results rule out the existence of long-range base-pairing interactions between loop C and either loop D or loop E. The data also confirm that loops D and E in the wild-type 5 S RNA adopt unusual secondary structures and illustrate the importance of nucleotide sequence in the formation of intrinsic local loop conformations via non-canonical base-pairs and specific base-phosphate contacts. Consistent with this conclusion is our observation that the domain 3 fragment of Xenopus oocyte 5 S rRNA adopts the same conformation as the corresponding region in the full-length 5 S rRNA.

Constitutive transactivation by the thyroid hormone receptor and a novel pattern of activity of its oncogenic homolog v-ErbA in Xenopus oocytes.

In Xenopus oocytes, the rat thyroid hormone receptor alpha (rTR alpha), but not its oncogenic homolog v-ErbA, constitutively activated thyroid hormone (T3)-responsive reporter genes at four positive thyroid hormone-responsive elements (TREs). At a subset of the positive TREs tested, the addition of T3 resulted in a further enhancement of reporter gene activation. In contrast, both rTR alpha and v-ErbA functioned as constitutive activators when bound to the clone 122 TREs, which are induced by unliganded TR in mammalian cells. Therefore, the responses of the ligand-independent activation domains of TR and v-ErbA to cell-specific and TRE-mediated induction are not equivalent. Coexpression of the human retinoid X receptor alpha (hRXR alpha) enhanced both ligand-dependent and ligand-independent activation functions of rTR alpha and human TR beta (hTR beta) at a palindromic TRE (TREp). An endogenous TR activity mediated T3 induction of TREp, while being repressed by an in vitro-generated dominant negative mutant of TR. T3-mediated gene activation, by exogenous or endogenous TR, was repressed by v-ErbA at three positive TREs, but not at the TRE from the third intron of the rat GH gene (rGH3 TRE). Interestingly, preinjection of nuclear protein extract from anterior pituitary cells converted v-ErbA into a constitutive activator at rGH3 TRE. The pituitary-specific factor Pit-1/GHF-1 or hRXR alpha did not induce activation by v-ErbA at rGH3 TRE, suggesting that the dominant negative phenotype of v-ErbA can be abolished by direct or indirect interactions with other nuclear factors.

The effects of P-box substitutions in thyroid hormone receptor on DNA binding specificity.

Three "P-box" amino acids within the DNA recognition alpha-helix of members of the steroid hormone and thyroid hormone families of nuclear receptors are known to determine the identity of two of the six base pairs within the half-sites of cognate DNA elements. We introduced P-box substitutions derived from different members of the thyroid hormone/estrogen receptor (T3R/ER) family into the beta-isoform of human thyroid hormone receptor (hT3R beta) and tested the DNA binding and transactivation activities of these mutants using thyroid hormone response elements (TREs) with half-sites composed of different sequences and arranged in different orientations. Different P-box sequences derived from the T3R/ER family resulted in distinct DNA binding specificities determined by the fourth base pair of the half-site. Thyroid hormone receptor mutants containing EGA, EAA, EGS substitutions for the wild type EGG P-box bound with wild type affinity to consensus AGGTCA half-sites, regardless of orientation. TREs composed of AGGACA half-sites bound hT3R beta s with an EGG or EAA P-box sequence, but not those with EGA or EGS P-box sequence. A reversal of this specificity was observed on a direct repeat TRE with AGGGCA half-sites. Additionally, an ESG P-box substitution in hT3R beta prevented the receptor from binding to a direct repeat as a homodimer, but this mutant could bind as a heterodimer with retinoid X receptor or to the everted repeat TRE from the chicken lysozyme promoter.

Functional analysis of the amino acids in the DNA recognition alpha-helix of the human thyroid hormone receptor.

The roles in DNA binding and transcriptional activation of individual amino acids in the putative recognition alpha-helix of the first zinc finger of the beta-isoform of the human thyroid hormone receptor (hT3R beta) have been probed by site-directed mutagenesis. Alanine substitutions of highly conserved residues involved in the folding of this zinc finger abolished the binding of hT3R beta to various thyroid response elements. A similar effect was observed for alanine substitutions of those conserved residues in hT3R beta that were expected to make specific contacts to DNA bases common to all hormone response elements. The three P-box amino acids have previously been shown to be essential for discrimination of the base pairs that differ between the DNA binding sites for related steroid/thyroid hormone receptors. In hT3R beta, the P-box residues are E, G, and G; the results of this study show that alanine substitution of the glutamic acid dramatically reduces DNA binding activity by hT3R beta, while the substitution of either glycine has little or no effect on DNA binding. The effects of alanine substitutions on hT3R beta transcriptional activation properties were consistent with the effect of these substitutions on DNA binding properties, with the exception of the second P-box amino acid. T3R beta substituted with alanine at this position is substantially more defective in transcriptional activation than it is in specific DNA binding. These results indicate that there are two separate mechanisms of response element discrimination by P-box amino acids of steroid/thyroid hormone receptors, one which operates at the level of DNA recognition and a second which operates at the level of transcriptional activation.

Ribosomal 5S RNA from Xenopus laevis oocytes: conformation and interaction with transcription factor IIIA.

This review describes extensive studies on 5S rRNA from X laevis oocytes combining conformational analyses in solution (using a variety of chemical and enzymatic probes), computer modeling, site-directed mutagenesis, crosslinking and TFIIIA binding. The proposed 3-dimensional model adopts a Y-shaped structure with no tertiary interactions between the different domains of the RNA. The conserved nucleotides are not crucial for the tertiary folding but they maintain an intrinsic structure in the loop regions. The model was tested by the analysis of several 5S rRNA mutants. A series of 5S RNA mutants with defined block sequence changes in regions corresponding to each of the loop regions was constructed by in vitro transcription of the mutated genes. Our results show that none of the mutations perturbs the Y-shaped structure of the RNA, although they induce conformational changes restricted to the mutated regions. The interaction of the resulting 5S rRNA mutants with TFIIIA was determined by a direct binding assay. Only the mutations in the hinge region between the 3 helical domains have a significant effect on the binding for the protein. Finally, TFIIIA was crosslinked by the use of trans-diamminedichloroplatinum (II) to a region covering the fork region. Our results show that (i) the tertiary structure does not involve long-range interactions; (ii) the intrinsic structures in loops are strictly sequence-dependent; (iii) the hinge nucleotides govern the relative orientation of the 3 helical domains; (iv) TFIIIA recognizes essentially specific features of the tertiary structure of 5S rRNA.

Photobiological properties of a novel, naturally occurring furoisocoumarin, coriandrin.

The photobiological properties of a novel, naturally occurring furoisocoumarin isolated from coriander and named coriandrin are described. Photosensitized lethal and mutagenic effects in bacteria indicate that it is more active than psoralen. It is a weak frameshift mutagen in the dark. Mammalian cells in tissue culture are photosensitized more actively with coriandrin than with psoralen even though preliminary evidence from interrupted radiation experiments and DNA analysis suggest that coriandrin does not form DNA interstrand crosslinks. Sister chromatid exchanges were induced with a unit dose of 1.1 x 10(-2) with coriandrin; the value for psoralen is 3 x 10(-3). Coriandrin appears to be metabolized more rapidly than furocoumarins by liver mixed function oxidases. Skin photosensitizing activity is very weak compared with psoralen, a surprising observation considering its potency in biological test systems.

Interaction of R17 coat protein with its RNA binding site for translational repression.

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kd at 2 degrees C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of Ka and a delta H = -19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RNAs failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific. We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.

Characterization of the RNA binding properties of transcription factor IIIA of Xenopus laevis oocytes.

A nitrocellulose filter binding assay has been developed to study the interaction of Xenopus transcription factor IIIA with 5S RNA. The protein binds Xenopus oocyte 5S RNA with an association constant of 1.4 X 10(9) M-1 at 0.1 M salt, pH 7.5 at 20 degrees C. TF IIIA binds wheat germ 5S RNA with a two-fold higher affinity, E. coli 5S RNA with a four-fold weaker affinity, and has a barely detectable interaction with yeast tRNAphe. The preference for binding eukaryotic 5S RNA is enhanced in competition assays. The homologous reconstituted complex contains one molecule each of protein and 5S RNA and is indistinguishable from native 7S RNP in mobility on non-denaturing polyacrylamide gels. The conformation of the RNA in reconstituted particles is identical to the conformation of RNA in native 7S RNP. Further analysis of the homologous interaction reveals that complex formation is a favoured both by enthalpy and entropy. The 5S RNA binding activity has a broad pH optimum spanning pH 6.0 to pH 8.0. Determination of the salt dependence of Ka reveals that as many as 5 lysine-phosphate type ionic bonds may be formed in the homologous complex. Approximately 68% of the free energy of complex formation is contributed by non-electrostatic interactions between TF IIIA and Xenopus 5S RNA.

The role of highly conserved single-stranded nucleotides of Xenopus 5S RNA in the binding of transcription factor IIIA.

The role of highly conserved single-stranded sequence elements of Xenopus 5S RNA in the binding of transcription factor IIIA (TFIIIA) was studied. A series of mutant 5S RNA genes were constructed with defined block sequence changes in regions corresponding to each of the single-stranded loops of the transcribed 5S RNA. The interaction of the resulting mutant 5S RNA molecules with TFIIIA was determined both by a direct binding assay and by a competition assay. With one exception, the substitution of highly conserved single-stranded loop sequences had only a modest effect on the binding of TFIIIA. The single exception was loop A, which ironically is not part of the protected site of TFIIIA on 5S RNA. The possible involvement of loop A in the coaxial stacking of the helical domains of 5S RNA, and how this might affect TFIIIA binding, are discussed.

Characterization of the equilibrium binding of Xenopus transcription factor IIIA to the 5 S RNA gene.

A nitrocellulose filter-binding assay has been developed to study the interaction of Xenopus transcription factor IIIA (TFIIIA) with its specific binding site on the 5 S RNA gene. The protein binds to a DNA restriction fragment containing a Xenopus oocyte 5 S RNA gene (5 S DNA) with an apparent association constant of 1.90 x 10(9) M-1 in 0.1 M salt, pH 7.5, at 22 degrees C. Under these assay conditions, the protein has approximately a 100-fold lower binding affinity for DNA fragments that do not contain a 5 S RNA gene. Analysis of the temperature dependence of the binding of TFIIIA to 5 S DNA indicates that the interaction is largely enthalpy driven at temperatures above 19 degrees C, while it is largely entropy driven at lower temperatures. One molecule of TFIIIA binds per 5 S RNA gene, and this bimolecular complex dissociates with first order kinetics, having a half-life of 15.6 min. The DNA binding activity of the protein exhibits a broad pH optimum from 6.0 to 8.0, and is optimal at 5 mM MgCl2 decreasing rapidly at higher divalent ion concentrations. The specific binding of TFIIIA to 5 S DNA is insensitive to the identity of the monovalent cation present in the binding buffer. In comparison, the anion effects on DNA binding are dramatic, with a 100-fold decrease in binding affinity observed to follow the lyotropic series. This result suggests that there are several specific anion-binding sites on TFIIIA. Determination of the monovalent salt dependence of the association constant revealed that as many as 8 lysine-phosphate type ionic bonds are formed in the TFIIIA-DNA complex.

A difference in the importance of bulged nucleotides and their parent base pairs in the binding of transcription factor IIIA to Xenopus 5S RNA and 5S RNA genes.

Individual bulge loops present in Xenopus 5S RNA (positions 49A-A50 in helix III, C63 in helix II and A83 in helix IV), were deleted by site directed mutagenesis. The interaction of these mutant 5S RNA molecules with TFIIIA was measured by a direct binding assay and a competition assay. The results of these experiments show that none of the bulged nucleotides in Xenopus 5S RNA are required for the binding of TFIIIA. The affinity of the mutant 5S RNA genes for TFIIIA was also studied by a filter binding assay. In contrast to the effect that deleting bulged nucleotides had on the TFIIIA-RNA binding affinity, deletion of the corresponding A-T base pair at position +83 in 5S DNA was found to reduce the apparent association constant of TFIIIA by a factor of four-fold.