Contribution of individual amino acids to the nucleic acid binding activities of the Xenopus zinc finger proteins TFIIIIA and p43.

Xenopus transcription factor IIIIA (TFIIIA) binds to both 5S RNA and the 5S RNA gene in immature oocytes, an interaction mediated by nine zinc fingers. To determine the role of the central zinc fingers of the protein in these nucleic acid binding activities, a series of substitution mutants of TFIIIA were constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for DNA and RNA binding activities using a nitrocellulose filter binding assay. Finger 5, but not finger 4, 6, or 7, is involved in sequence-specific binding to the 5S RNA gene. A TWT amino acid motif in finger 6 makes a significant contribution to the binding of TFIIIA to 5S RNA, while mutations in fingers 4, 5, and 7 have little or no effect on RNA binding by TFIIIA. In striking contrast, a TWT motif in finger 6 of p43, another Xenopus zinc finger protein that binds to 5S RNA, is not necessary for 5S RNA binding by this protein. Evidence for the presence of inhibitory finger-finger interactions that limit the nucleic acid binding properties of individual zinc fingers within TFIIIA and p43 is discussed.

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1.

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.

Ornithine decarboxylase is a transcriptional target of tumor suppressor WT1.

The product of the Wilm's tumor suppressor gene, WT1, is a zinc-finger DNA-binding protein, which is thought to be a transcription factor. Two genes, those encoding epidermal growth factor receptor and syndecan-1, are known to be endogenous targets of WT1. Previous studies had identified binding sites for WT1 in the promoter of the ornithine decarboxylase (ODC) gene. In this paper, we tested whether the endogenous ODC gene might be a target of WT1 by establishing lines of baby hamster kidney (BHK) cells that expressed WT1 isoform A under control of a tetracycline-regulated expression system. When expression of WT1 was activated in BHK cells, the cellular level of ODC mRNA declined, with kinetics that correlated with the increase in WT1 level, demonstrating that the endogenous ODC gene was indeed responsive to cellular level of WT1. WT1 isoforms A and B inhibited the activity of the ODC promoter by approximately fivefold in transiently transfected BHK cells, while isoforms C and D, which have altered DNA binding domains, had no significant effect. The sequence CTCCCCCGC, located at nucleotides -106 to -98 relative to the site of transcriptional initiation in the ODC gene, interacted with the zinc-finger domain of isoforms A and B of WT1 with high affinity and specificity. A mutation in the binding site that disrupted this interaction partially removed the inhibition of ODC promoter activity by WT1, as did mutation of the two E-box sequences in intron I of the ODC gene. Simultaneous mutation of the WT1-binding motif and the two E-boxes completely abolished inhibition by WT1 of ODC promoter activity. These results, taken together, implicate the ODC gene as a downstream target of the tumor suppressor WT1.

Comparison of the DNA binding characteristics of the related zinc finger proteins WT1 and EGR1.

The interactions of the related zinc finger proteins WT1 and EGR1 with DNA have been investigated using a quantitative binding assay. A recombinant peptide containing the four zinc fingers of WT1 binds to the dodecamer DNA sequence GCG-TGG-GCG-TGT with an apparent dissociation constant (Kd) of (1.14 +/- 0.09) x 10(-9) M under conditions of 0.1 M KCl, pH 7.5, at 22 degrees C. Under the same conditions, a recombinant peptide containing the three zinc fingers of EGR1 binds to the dodecamer sequence, the first nine bases comprising the EGR consensus binding site, with an apparent Kd of (3.55 +/- 0.24) x 10(-9) M. The nature of the equilibrium binding of each peptide to DNA was investigated as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1 with DNA is an entropy-driven process, while the formation of the EGR1-DNA complex is favored by enthalpy and entropy. The DNA binding activities of both proteins have broad pH optima centered at pH 8.0. The binding of both proteins to DNA shows similar sensitivity to ionic strength, with approximately 7.7 +/- 0.8 ion pairs formed in the EGR1-DNA complex and 9.2 +/- 1.8 ion pairs formed in the WT1-DNA complex. Results of measuring the effects of point mutations in the DNA binding site on the affinity of WT1 and EGR1 indicates a significant difference in the optimal binding sites: for EGR1, the highest affinity binding site has the sequence GNG-(T/G)GG-G(T/C)G, while for WT1 the highest affinity binding site has the sequence G(T/C)G-(T/G)GG-GAG-(T/C)G(T/C).

Effects of Denys-Drash syndrome point mutations on the DNA binding activity of the Wilms' tumor suppressor protein WT1.

A number of point mutations in the zinc finger domain of the Wilms' tumor suppressor protein WT1 have been isolated from the DNA of patients with Denys-Drash syndrome, an association of Wilms' tumor, nephropathy, and genital anomalies. To date, five different mutations that alter amino acids predicted to interact specifically with nucleotides in the target DNA sequence have been described. Two of these mutations are located in zinc finger 2 (R366H, R366C), and three are located in finger 3 (R394W, D396G, D396N). These five Denys-Drash mutations were introduced into WT1-ZFP, a recombinant polypeptide containing the zinc finger domain of WT1, and the effects of these mutations on DNA sequence specificity were determined using a selection, amplification, and binding (SAAB) assay. The SAAB assay was carried out using two different DNA templates, one with a randomized finger 2 subsite (GCG TGG NNN TGT) and one with a randomized finger 3 subsite (GCG NNN GCG TGT). A comparison of the DNA sequences selected by WT1-ZFP and by Denys-Drash mutants suggests that the point mutations reduce the sequence selectivity of the zinc finger protein. With the exception of the R394W mutant, the other Denys-Drash mutations selected one alternative sequence in addition to the wild-type DNA subsite sequence. The binding affinities of these proteins for their selected sequences were determined using a quantitative nitrocellulose filter binding assay. These results revealed that the wild-type WT1 binds with slightly higher affinity to sequences with GAG in the finger 2 subsite than sequences with the EGR-1 consensus GCG finger 2 subsite. With the exception of R394W, which appears to lack specific DNA binding activity, the Denys-Drash mutants bound to selected DNAs with 1.4-14-fold lower affinities than the wild-type WT1-ZFP. These results suggest that the clinical phenotype of Denys-Drash syndrome can be associated with a modest reduction in the DNA binding affinity of WT1.

Retinoid X receptor alters the determination of DNA binding specificity by the P-box amino acids of the thyroid hormone receptor.

Nuclear hormone receptors bind to hormone response elements in DNA consisting of two half-sites of 6 base pairs. The P-box amino acids of each receptor determine the identities of the central nucleotides of the half-site. 57 P-box variants of the human thyroid hormone receptor (hT3Rbeta) were used to demonstrate the relationship between P-box sequence and DNA binding specificity by homodimers and heterodimers formed with the retinoid X receptor (RXR). In general, the formation of heterodimers relieved many of the constraints on the compatibility of hT3Rbeta P-box sequences with DNA binding. Effects were most dramatic for heterodimers bound to a direct repeat spaced by four base pairs. RXR also overrides the P-box-derived DNA binding specificity of hT3Rbeta when heterodimers are bound to inverted or everted repeat elements. These effects of RXR are most pronounced on AGGTCA half-sites but are squelched when the RXR partner of the heterodimer is bound to an AGGACA half-site. The influence of RXR on hT3Rbeta DNA binding specificity varies with the orientation of half-sites in the element, the identity of the fourth base pair of the half-site, and the spacing between the half-sites of direct repeats. These differences suggest that the DNA binding domains of RXR-hT3Rbeta heterodimers are not positioned equivalently on the various elements, affecting the manner in which the P-box amino acids of hT3Rbeta interact with base pairs within the half-site.

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.

Effects of zinc finger mutations on the nucleic acid binding activities of Xenopus transcription factor IIIA.

Transcription factor IIIA (TFIIIA) is required for the activation of 5S RNA gene transcription as well as the storage of 5s RNA as a 7S ribonucleoprotein particle. Interaction with both nucleic acids is mediated through nine C2H2 zinc fingers. In order to determine amino acid regions necessary for nucleic acid interaction, a series of substitution mutants Xenopus laevis TFIIIA have been constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for 5S RNA gene and 5S RNA binding activities using a nitrocellulose filter binding assay. All of the mutant TFIIIA proteins retained full 5S RNA binding activity. Substitution of fingers 2, 3, and 4-6 of TFIIIA with zinc finger sequences from other proteins significantly reduced the interaction of the protein with the 5S RNA gene. In contrast, substitution of finger 1 or finger 7 had little effect on the interaction of TFIIIA with the 5S RNA gene. The results of scanning substitution mutagenesis within the first three zinc fingers of TFIIIA suggested that DNA contacts made by the alpha-helical regions of finger 2 and particularly of finger 3 provide the majority of the free energy of the TFIIIA-DNA interaction. Basic amino acids found at the same position within the alpha-helices of fingers 2 and 3 of TFIIIA are required for high-affinity DNA binding activity. The identification of amino acid residues critical for the formation of a TFIIIA-DNA complex contributes to our understanding of zinc finger protein-nucleic acid interactions.

Relationship between P-box amino acid sequence and DNA binding specificity of the thyroid hormone receptor. The effects of half-site sequence in everted repeats.

The three P-box amino acids in the DNA recognition alpha-helix of steroid/thyroid hormone receptors participate in the discrimination of the central base pairs of the hexameric half-sites of receptor response elements in DNA. A series of 57 variants of the beta isoform of the human thyroid hormone receptor were constructed in which the 19 possible amino acid substitutions were incorporated at each of the three P-box positions. The effects of these substitutions on the sequence specificity of the DNA binding activity of the receptor were analyzed using 16 everted repeat elements which differed in sequence in the two central base pairs of the hexameric half-sites. Only receptors with glutamate or aspartate as the first P-box amino acid had detectable DNA binding affinity on everted repeats with AGGNCA half-sites. Only those receptors with alanine, glycine, serine, or proline in the second P-box position were able to bind to this same group of everted repeat elements. In contrast, many of the variant receptors with substitutions at the third P-box position were capable of binding to the AGGNCA group of repeat elements. The actual substitutions at the third P-box position that were compatible with binding depended upon the identity of the fourth base pair of the AGGNCA half-sites. Of the remaining 12 everted repeat sequences, only those with AGTTCA or AGTCCA half-sites were able to bind any of the receptors. In addition to wild type receptor, several variant receptors with amino acid substitutions in either the first or third P-box position were able to bind to the everted repeat with AGTTCA half-sites. The everted repeat with AGTCCA half-sites was bound by receptors with a DGG, NGG, or EGQ P-box sequence, but not the wild type receptor which has an EGG P-box sequence. These data demonstrate that all three P-box positions of the thyroid hormone receptor function to discriminate between half-sites that differ in sequence at the third and fourth base pairs.

Relationship between P-box amino acid sequence and DNA binding specificity of the thyroid hormone receptor. The effects of sequences flanking half-sites in thyroid hormone response elements.

The three P-box amino acids in the DNA recognition alpha-helix of steroid/thyroid hormone receptors participate in the discrimination of the central base pairs of the hexameric half-sites of receptor response elements in DNA. Using a series of variant receptors incorporating all 19 possible substitutions for each individual P-box amino acid of the human thyroid hormone receptor (hT3R beta), we demonstrated that the first P-box position must have a glutamate, and the second P-box position must have either an alanine or a glycine for high affinity binding to everted repeat elements with half-site sequences of AGGNCA. In the present study, the influence of half-site flanking sequence on the compatibility of P-box amino acids in hT3R beta with DNA binding was investigated. When a 5' sequence of CTG flanked AGGNCA half-sites in an everted repeat, several additional P-box variant receptors were able to bind to the DNA that were not able to bind when the half-sites were flanked with the 5' sequence CAG. Flanking sequence had the most dramatic effects on amino acid substitutions at the first P-box position, with smaller effects observed at the second P-box position and only subtle effects observed at the third P-box position. Expansion of the number of P-box sequences compatible with binding of hT3R beta to thyroid hormone response elements required the thymidine in the CTG flanking sequence, an everted repeat of the AGGNCA half-sites, and an intermolecular interaction in the C terminus of the receptor.

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.

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.

High affinity binding sites for the Wilms' tumour suppressor protein WT1.

The Wilms' tumour suppressor protein (WT1) is a putative transcriptional regulatory protein with four zinc fingers, the last three of which have extensive sequence homology to the early growth response-1 (EGR-1) protein. Although a peptide encoding the zinc finger domain of WT1[-KTS] can bind to a consensus 9 bp EGR-1 binding site, current knowledge about the mechanisms of zinc finger-DNA interactions would predict a more extended recognition site for WT1. Using a WT1[-KTS] zinc finger peptide (WT1-ZFP) and the template oligonucleotide GCG-TGG-GCG-NNNNN in a binding site selection assay, we have determined that the highest affinity binding sites for WT1[-KTS] consist of a 12 bp sequence GCG-TGG-GCG-(T/G)(G/A/T)(T/G). The binding of WT1-ZFP to a number of the selected sequences was measured by a quantitative nitrocellulose filter binding assay, and the results demonstrated that these sequences have a 4-fold higher affinity for the protein than the nonselected sequence GCG-TGG-GCG-CCC. The full length WT1 protein regulates transcription of reporter genes linked to these high affinity sequences. A peptide lacking the first zinc finger of WT1[-KTS], but containing the three zinc fingers homologous to EGR-1 failed to select any specific sequences downstream of the GCG-TGG-GCG consensus sequence in the binding site selection assay. DNA sequences in the fetal promoter of the insulin-like growth factor II gene that confer WT1 responsiveness in a transient transfection assay bind to the WT1-ZFP with affinities that vary according to the number of consensus bases each sequence possesses in the finger 1 subsite.

Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp.

The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.

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.

Contribution of individual base pairs to the interaction of TFIIIA with the Xenopus 5S RNA gene.

The effects of a series of point mutations within the Xenopus borealis somatic-type 5S RNA gene on transcription factor IIIA (TFIIIA) binding affinity were quantified. These data define a critical sequence-dependent contact region within the classical box C promoter element from base pair 80 to 91. Substitution of GC base pairs at positions 81, 85, 86, 89, and 91 significantly reduce TFIIIA binding affinity. Base pairs located at other positions within the box C contact region provide a moderate contribution to TFIIIA-5S gene interaction. In contrast to the extensive set of sequence contacts within the box C element, TFIIIA interaction is localized primarily to two GC base pairs at positions 70 and 71 within the intermediate promoter element. A selected amplification and binding assay (SAAB) was performed with a synthetic internal control region (ICR) randomized from base pair 78 to 95 to identify box C promoter sequences bound with high affinity by TFIIIA. The wild-type 5S RNA gene sequence from 79 to 92 is strongly selected. These results are consistent with the critical role of the box C element in sequence-dependent promoter recognition by TFIIIA.

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.

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.

Interaction of Xenopus TFIIIC with the TFIIIA.5 S RNA gene complex.

The general transcription factor TFIIIC is necessary for transcription initiation by RNA polymerase III. TFIIIC binds predominantly to the B-Block promoter element, which is present in tRNA genes, several viral RNA genes and repetitive DNA elements, and to the TFIIIA.DNA complex on 5 S RNA genes. Here we report a characterization of Xenopus laevis TFIIIC and its interaction with the TFIIIA.5 S RNA gene complex. A polypeptide with apparent molecular mass of 85 kDa was specifically cross-linked to a B-Block oligonucleotide by UV light. This polypeptide was present in the partially purified TFIIIC fraction and in a complex with a B-Block double-stranded oligonucleotide isolated by nondenaturing gel electrophoresis. TFIIIC.TFIIIA.DNA gel mobility shift complexes were obtained using B-Block DNA affinity-purified TFIIIC and buffer conditions employing low Mg2+ (1 mM) and high dithiothreitol (7 mM) concentrations. Three TFIIIC.TFIIIA.5 S RNA gene complexes were observed by gel mobility shift analysis. One of these complexes was resistant to dissociation by the addition of competing DNA, but the formation of all three complexes was prevented by the inclusion of excess specific competitor DNA in the initial binding reactions. The apparent affinity of TFIIIC for the TFIIIA.5 S DNA complex was 5-fold higher for the somatic-type 5 S RNA gene than for the oocyte-type 5 S RNA gene. Mutations near the 5' boundary of the TFIIIA binding site alter the DNase I footprint of the TFIIIA.DNA complex and reduce the affinity of TFIIIA-mutant 5 S gene complexes for TFIIIC. Differences in TFIIIC affinity for the two classes of 5 S RNA genes may play a role in the developmental regulation of these gene families.

Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents.

Helical organisms with novel ultrastructural characteristics were isolated from the intestinal mucosa of rats and mice. These bacteria were characterized by the presence of 9 to 11 periplasmic fibers which appeared as concentric helical ridges on the surface of each cell. The cells were motile with a rapid corkscrewlike motion and had bipolar tufts of 10 to 14 sheathed flagella. The bacteria were microaerophilic, nutritionally fastidious, and physiologically similar to Helicobacter species and Wolinella succinogenes but could be differentiated from these organisms by their unique cellular ultrastructure. Using 16S rRNA sequencing, we found that strain ST1T (T = type strain) was related to previously described Helicobacter species, "Flexispira rappini," and W. succinogenes. The closest relatives of strain ST1T were Helicobacter mustelae and "F. rappini" (average similarity value, 96%). On the basis of phylogenetic data, strain ST1T (= ATCC 49282T) represents a new species of the genus Helicobacter, for which we propose the name Helicobacter muridarum.