TFIIIA Binding Research Articles

Zinc(II) ions selectively interact with DNA sequences present at the TFIIIA binding site of the Xenopus 5S-RNA gene.

It has been known for some time that zinc, as well as most transition metal ions, is capable of binding to the DNA bases. However, little is known about the presence and distribution of metal binding sites along naturally occurring genomic DNA molecules. In this paper, the interaction of zinc with the Xenopus 5S-RNA gene has been studied and several metal binding sites have been identified on the basis of the changes in chemical reactivity observed in the presence of the metal. The strongest zinc-binding sites of the Xenopus 5S-RNA gene correspond to GGG trinucleotide repeats. Some GG dinucleotides also show a significant affinity for zinc. Interestingly, the binding site for TFIIIA, a zinc-finger transcription factor, contains several sites with strong zinc affinity. In particular, a TGGGA sequence which is essential for the binding of TFIIIA shows the strongest affinity for zinc. The conformational properties of this DNA sequence, together with the high electronegative potential of GGG runs, is likely to determine its strong affinity for zinc. The possible biological relevance of these results is discussed.

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.

Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.

Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.

Histone-DNA contacts in a nucleosome core containing a Xenopus 5S rRNA gene.

We describe histone-DNA cross-linking in a nucleosome core containing a Xenopus borealis somatic 5S rRNA gene. Histones H3 and H4 are cross-linked to DNA within 30 bp to either side of the dyad axis. Histones H2A/H2B and H3 are cross-linked to DNA where it enters and exists, wrapping around the histone octamer. These latter interactions extend for 80 bp to one side of the dyad axis of the nucleosome core, including the entire binding site for transcription factor TFIIIA. These extensive interactions with linker DNA might account for inhibition of TFIIIA binding and also might assist in the folding of internucleosomal DNA within the chromatin fiber.

The conformation of loop E of eukaryotic 5S ribosomal RNA.

The solution structure of a 27-nucleotide duplex, including the internal loop E from Xenopus laevis 5S ribosomal RNA, has been studied by two-dimensional NMR spectroscopy, followed by restrained molecular dynamics. The highly conserved internal loop closes to form a G.A base pair and a reverse-Hoogsteen A.U base pair. Extensive interstrand stacking between these uncommon base pairs provides a structural explanation for an interstrand ultraviolet-induced cross-link. A guanosine residue is bulged into the major groove and may form a base-triple with the adjacent reverse-Hoogsteen A.U pair. The structure of the less highly-conserved portion of the loop is less well-defined by the NMR data. A single-nucleotide deletion mutant has a very different, open conformation without mismatched base pairs [Varani, G., Wimberly, B., & Tinoco, I. Jr. (1989) Biochemistry 28, 7760-7772]. The implications of the structure for binding of the transcription factor TFIIIA and the cytotoxin alpha-sarcin are discussed.

A positive role for histone acetylation in transcription factor access to nucleosomal DNA

Acetylation of the N-terminal tails of the core histones directly facilitates the recognition by TFIIIA of the 5S RNA gene within model chromatin templates. This effect is independent of a reduction in the extent of histone-DNA interactions or a change in DNA helical repeat; it is also independent of whether a histone tetramer or octamer inhibits TFIIIA binding. Removal of the N-terminal tails from the core histones also facilitates the association of TFIIIA with nucleosomal templates. We suggest that the histone tails have a major role in restricting transcription factor access to DNA and that their acetylation releases this restriction by directing dissociation of the tails from DNA and/or inducing a change in DNA configuration on the histone core to allow transcription factor binding. Acetylation of core histones might be expected to exert a major influence on the accessibility of chromatin to regulatory molecules.

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.

Binding of TFIIIA to derivatives of 5S RNA containing sequence substitutions or deletions defines a minimal TFIIIA binding site.

The repetitive zinc finger domain of transcription factor IIIA binds 5S DNA and 5S RNA with similar affinity. Site directed mutagenesis of the Xenopus borealis somatic 5S RNA gene has been used to produce a series of derivatives of 5S RNA containing local sequence substitutions or sequence deletions. Gel mobility shift analyses of the binding of TFIIIA to these altered 5S RNAs revealed that all three of the helical stems of the 5S RNA secondary structure are required for binding. TFIIIA was observed to bind with normal affinity to RNAs lacking 12 nucleotides at either the loop c or loop e/helix V regions of 5S RNA, as well as to a double mutant containing both deletions. The secondary structure of the resulting 96-nucleotide RNA, studied using structure-specific ribonucleases, was found to resemble the central portion of 5S RNA.

Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'-deoxyuridine-substituted DNA.

A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.

Mutations in 5S DNA and 5S RNA have different effects on the binding of Xenopus transcription factor IIIA

The effects on TFIIIA binding affinity of a series of substitution mutations in the Xenopus laevis oocyte 5S RNA gene were quantified. These data indicate that TFIIIA binds specifically to 5S DNA by forming sequence-specific contacts with three discrete sites located within the classical A and C boxes and the intermediate element of the internal control region. Substitution of the nucleotide sequence at any of the three sites significantly reduces TFIIIA binding affinity, with a 100-fold reduction observed for substitutions in the box C subregion. These results are consistent with a direct interaction of TFIIIA with specific base pairs within the major groove of the DNA. A comparison of the TFIIIA binding data for the same mutations expressed in 5S RNA indicates that the protein does not make any strong sequence-specific contacts with the RNA. Although the protein footprinting sites on the 5S DNA and 5S RNA are coincident, nucleotide substitutions in 5S RNA which moderately reduce TFIIIA binding affinity do not correspond at all to the three specific TFIIIA interaction sites within the gene. The implications of these results for models which attempt to reconcile the DNA and RNA binding activities of TFIIIA by proposing a common structural motif for the two nucleic acids are discussed.

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.

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 inthe 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.

Identification of a TFIIIa binding site on the 5′ flanking region of the TFIIIA gene

Xenopus transcription factor IIIA (TFIIIA) regulates 5S ribosomal RNA gene transcription in a positive manner by binding to the internal control region (ICR) of the 5S RNA gene. The present study reports the identification of a TFIIIA binding site on the 5' flanking region of the Xenopus laevis gene that codes for the synthesis of this transcription factor. This TFIIIA binding site (deduced from TFIIIA-dependent alterations in DNase I protection patterns) extends on the 5' flanking region from about nucleotide -326 to -264 on the non-coding strand and from -331 to -271 on the coding strand. The affinity of TFIIIA for the 5' flanking region of its own gene is less than that for the 5S RNA gene but within the same order of magnitude. A sequence similarity between this newly identified binding site and the 5S gene ICR is the presence of purine-rich tracts. EDTA chelation of TFIIIA inhibits binding to this 5' flanking element indicating, as with the 5S RNA gene, zinc is required for DNA binding specificity. When TFIIIA is bound to 5S RNA in the 7S particle, the protein does not bind to this 5' flanking region, an observation similar to that observed with binding to the 5S RNA gene. These results indicate TFIIIA is using similar nucleic acid binding domain(s) for interaction with the 5S gene, 5S RNA, and the 5' flanking region of its own gene. This upstream DNA region to which TFIIIA binds has been previously shown to contain a negative and a positive regulatory element for transcription of the TFIIIA gene (Scotto, K.W., Kaulen, H., and Roeder, R.G., 1989, Genes & Develop. 3, 651-662). The present results indicate TFIIIA binds to the negative control element located at -306 to -289 and possibly interacts/interferes with another transcription factor which binds to the positive control element extending from -271 to -253.

Zinc release from Xenopus transcription factor IIIA induced by chemical modifications.

Xenopus transcription factor IIIA (TFIIIA) contains two tightly bound intrinsic Zn2+ ions that are released through treatment with either p-(hydroxymercuri)benzenesulfonate (PMPS) or diethyl pyrocarbonate (DEP) as monitored by the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The inactivation of TFIIIA by DEP as detected by an in vitro 5S RNA gene transcription assay was correlated with the extent of modification of histidine residues and Zn2+ release. Following reaction with PMPS, the 7S particle was dissociated into free TFIIIA and 5S RNA. This dissociation could be correlated with the extent of modification of cysteine residues as well as the Zn2+ release. The dissociation of the 7S particle was reversed by the addition of excess thiol reagent. However, the reversibility could be inhibited by EDTA, suggesting that Zn2+ was required for the binding of TFIIIA to 5S RNA. In the presence of PMPS- or DEP-modified TFIIIA or Zn2+-depleted TFIIIA, the fluorescence emission maximum of the hydrophobic probe, 8-anilinonaphthalenesulfonate, was blue-shifted by 30 nm, while only less than a 10-nm blue shift was observed in the presence of either the 7S particle or TFIIIA. These results indicate that the two Zn2+ ions in TFIIIA are coordinated with the cysteine and histidine residues and are required for maintenance of the proper conformation of TFIIIA.

Transcription of 5S RNA genes containing insertion mutation and the assembly of preinitiation complexes

The transcription of Xenopus 5S RNA genes containing insertions in and around the intragenic control region has been analyzed in a HeLa cell-free system. A 15 bp insertion within the intragenic region greatly diminished binding of TFIIIA and concomitantly reduced binding of TFIIIB and transcription in vitro . This study helps to define more precisely the promoter organization of Xenopus 5S RNA genes for the efficient interactions with transcription factors.

Molecular organization of developmentally regulated Dictyostelium discoideum ubiquitin cDNAs

Dictyostelium discoideum ubiquitin mRNAs are regulated in a complex fashion during spore germination and multicellular development. Species of mRNA of 1900, 1400, 1100, 840, 580, and 500 nucleotides (nt) are found which are expressed differentially during different stages of development. DNA blot analysis indicates that ubiquitin genes constitute a multigene family of at least six genes. cDNAs representing all the ubiquitin mRNA transcripts were isolated and sequenced. The Dictyostelium mRNAs are organized as tandem repeats of the 76 amino acid ubiquitin unit (228 nt). We isolated one cDNA containing seven of these tandem repeats, and two different five- and three-repeat cDNAs. In addition, 2 cDNAs containing a single ubiquitin repeat fused at its 3' end to an unrelated 52 and 78 amino acid extension were identified. There is a remarkable similarity in the sequences of the non-ubiquitin extensions among yeast and mammalian counterparts. The extensions are very basic, containing approximately 30% lysine/arginine. Another common feature of these proteins is the presence of a common structural motif containing cysteine residues at conserved positions, suggesting a metal binding domain that matches a consensus sequence of Xenopus transcription factor TFIIIA and other nucleic acid binding proteins. The characterization of ubiquitin cDNAs and genomic sequences in D. discoideum now makes the understanding of its developmental regulation feasible.

A difference in the importance of bulged nucleotides and their parent base pairs in the binding of transcription factor IIIA toXenopus5S 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.

Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro.

An extract of whole oocytes (oocyte S150) differentially transcribes Xenopus oocyte and somatic 5S RNA genes. In the oocyte S150, transcription complexes with different stabilities are assembled onto oocyte and somatic 5S DNA. The stability of the transcription complex is dependent on activities present in a fraction containing transcription factor TFIIIC. This fraction stabilizes the binding of the positive transcription factor TFIIIA to a somatic 5S RNA gene much more efficiently than it does to an oocyte gene. The oocyte S150 transcription extract is deficient in TFIIIC such that supplementation with a fraction enriched in this transcription factor selectively stimulates oocyte 5S DNA transcription. Previously it has been shown that an egg extract deficient in TFIIIA selectively transcribes somatic 5S RNA genes. Thus under conditions where there is differential stability of transcription complexes, limitation of either TFIIIA or TFIIIC may exaggerate the differential expression of two genes.

TFIIIA binds with equal affinity to somatic and major oocyte 5S RNA genes.

Current models for the differential control of expression of Xenopus somatic and oocyte 5S RNA genes suggest that an impaired ability to bind TFIIIA contributes to the inactivation of oocyte 5S RNA genes in somatic cells. The somatic 5S RNA gene is transcribed more efficiently than the major oocyte 5S RNA gene in S-150 extracts of mature oocytes. However, this differential transcription efficiency is not determined simply by the relative affinity for binding of a positive transcription factor, TFIIIA. We have compared the abilities of somatic, major oocyte, and minor oocyte 5S RNA genes to bind TFIIIA using both a standard footprint competition assay and an indirect DNase protection assay. This indirect DNase protection assay permits the direct comparison of TFIIIA binding to two templates in one reaction. Both assay methods indicate that the major oocyte 5S RNA gene and the somatic 5S RNA gene bind TFIIIA with equal affinity. As a further control, we have confirmed earlier work indicating that the minor oocyte gene binds TFIIIA with a reduced affinity. Binding of TFIIIA to these three 5S RNA genes results in a different pattern of protection of each gene. We suggest that slight differences in the contacts between TFIIIA and the 5' border of the control region influence the ability of additional transcription factors to bind to the TFIIIA:5S DNA complex.