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At least 19 recordsLinked to original sources
PubMed1986

Structure of the gene for Xenopus transcription factor TFIIIA.

The eucaryotic transcription factor TFIIIA is required for 5S RNA transcription in Xenopus, and changes in the level of TFIIIA have been implicated in the differential expression of 5S RNA genes. In this paper, we report the isolation and sequencing of the X. laevis TFIIIA gene. The gene is approximately 11 kb in length and consists of 9 coding segments separated by 8 introns. A sequence of 30 amino acid residues is known to repeat imperfectly 9 times in tandem within the TFIIIA protein, and Miller et al. (EMBO J. 4, 1609-1614, 1985) proposed that TFIIIA evolved by duplication of a primordial 30 amino acid residue unit. Our results from DNA sequence analysis support their proposal by showing that some of the exon-intron boundaries correspond closely to the repeating unit. We also found that the 5' flanking sequence of the TFIIIA gene contains a TATA box (TATATAA) at position -32 and a CAAT box (GCCAATCC) at position -96 and that the site of polyadenylation is 255 residues 3' of the stop codon. Finally, we have shown that the coding sequence of the TFIIIA gene is significantly polymorphic.

Amino Acid Sequence↗
PubMed1989

Presence of multiple species of polypeptides immunologically related to transcription factor TFIIIA in adult Xenopus tissues.

Transcription of 5S RNA gene in Xenopus oocytes requires a 38 kDa transcription factor TFIIIA, which interacts with the 50 bp internal control region of the gene. We looked for TFIIIA-like polypeptides in the extracts of adult Xenopus tissues on the basis of their antigenic cross-reactivity to anti-TFIIIA antibody. Several species of polypeptides ranging from 30 to 50 kDa were found in kidney, stomach, liver and testis. Although these polypeptides reacted specifically to anti-TFIIIA antibody, proteolytic peptide mapping of three representative ones did not reveal any mutual similarities. They also seemed to be distinct from TFIIIA. Possible functions of these proteins are discussed.

Animals↗
PubMed1989

Crosslinking of transcription factor TFIIIA to ribosomal 5S RNA from X. laevis by trans-diamminedichloroplatinum (II).

Trans-diamminnedichloroplatinum (II) was used to induce reversible crosslinks between 5S rRNA and TFIIIA within the 7S RNP particle from X. laevis immature oocyte. The crosslinked fragments have been unambiguously identified. These fragments exclusively arise from three RNA regions centered around the hinge region at the junction of the three helical domains. Major crosslinking sites are located in region 9-21 (comprising loops A and helix II) and region 54-71 (comprising loop B, helices II and V). A minor site is also found in the 3' part of helix I and helix V (region 100-120). Our results point to the crucial role of the junction region and of the three-dimensional folding of the RNA in the recognition of the 5S rRNA by TFIIIA.

Animals↗
PubMed1985

The primary structure of transcription factor TFIIIA has 12 consecutive repeats.

Analysis of the amino acid sequence of transcription factor TFIIIA from Xenopus laevis reveals the presence of 12 repeating structures, each about 30 residues in length. These segments have been aligned and their secondary structure predicted. The repeats each contain two invariant cysteines and two invariant histidines, perhaps to coordinate a zinc cation. Possible nucleic acid interaction modes are discussed.

Amino Acid Sequence↗
PubMed2000

Energetics of the specific binding interaction of the first three zinc fingers of the transcription factor TFIIIA with its cognate DNA sequence.

The energetics of the specific interaction of a protein fragment (zf1-3) containing the three N-terminal zinc fingers of the Xenopus laevis transcription factor TFIIIA with its cognate DNA sequence, contained in a 15 bp DNA duplex were studied using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and fluorescence titration. The use of both ITC and DSC is necessary to provide values for the thermodynamic parameters that have been corrected for thermal fluctuations of the interacting molecules. In the temperature range from 13 degrees C to 45 degrees C (where all the binding reaction components are folded), formation of the complex is enthalpically driven with a negative heat capacity effect (DeltaC(p)). In this respect, the binding reaction of zf1-3 is similar to those of other proteins that bind in the major groove of DNA. It is dissimilar to the association reactions of proteins, however, that bind in the minor groove of DNA and that are driven by a dominating entropy factor. Comparison of the experimental values of DeltaH(ass) and DeltaC(p) with expected values of these parameters, calculated from the burial of polar and nonpolar molecular surfaces, indicates that the polar groups at the protein/DNA interface are not completely dehydrated upon formation of the complex. It also seems that the expected large positive entropy of dehydration upon forming the zfl-3/DNA complex ( approximately 1900 J * K(-1) * mol(-1)) cannot be balanced by the reduction in translational/rotational and configurational freedom of the protein to the level of the observed entropy of binding (38 J * K(-1) * mol(-1)). It is suggested that the additional negative entropy contribution comes from a damping of torsional motions in the DNA duplex.

Calorimetry, Differential Scanning↗
PubMed1992

The deduced sequence of the transcription factor TFIIIA from Saccharomyces cerevisiae reveals extensive divergence from Xenopus TFIIIA.

TFIIIA is an RNA polymerase III transcription factor that binds to the internal control region of the 5 S RNA gene as the first step in the assembly of a transcription complex. We have identified the gene encoding TFIIIA from Saccharomyces cerevisiae. Protein synthesized in vitro from the cloned gene has the same size, DNA-binding properties, and transcription factor activity as does purified yeast TFIIIA. Examination of the deduced sequence of the 50-kDa yeast transcription factor revealed the presence of nine zinc-finger motifs, a characteristic of Xenopus TFIIIA. Although the conservation of these nine putative DNA-binding domains is striking, the amino acid sequence throughout the corresponding fingers of the yeast and amphibian TFIIIAs has diverged extensively and in many instances the spacing between the residues that coordinate the zinc ions differs between the two proteins. A unique feature of the yeast protein is an 81-amino acid domain interrupting the repeated zinc-finger motifs between fingers 8 and 9. Additionally, the yeast and amphibian proteins differ in both the size and sequence of the amino- and carboxyl-terminal domains flanking the zinc fingers. The gene encoding yeast TFIIIA is present in single copy in the S. cerevisiae genome and is essential for cell viability. A carboxyl-terminal truncated form of the protein containing 4.5 zinc-finger motifs retains the ability to bind to DNA but is no longer active in promoting transcription in vitro.

Amino Acid Sequence↗
PubMedDate not supplied

Sequence homology of the yeast regulatory protein ADR1 with Xenopus transcription factor TFIIIA.

Classical yeast genetics coupled with the cloning of regulatory genes by complementation of function is a powerful means of identifying and isolating trans-acting regulatory elements. One such regulatory gene is ADR1 which encodes a protein required for transcriptional activation of the glucose-repressible alcohol dehydrogenase (ADH2) gene. We now report the nucleotide sequence of ADR1; it encodes a polypeptide chain of 1,323 amino acids, of which the amino-terminal 302 amino acids are sufficient to stimulate ADH2 transcription. This active amino-terminal region shows amino-acid sequence homology with the repetitive DNA-binding domain of TFIIIA, an RNA polymerase III transcription factor of Xenopus laevis. Similar domains are found in proteins encoded at the Krüppel and Serendipity loci of Drosophila melanogaster. We discuss the implications of this structural homology and suggest that a similar domain may exist in other yeast regulatory proteins such as those encoded by GAL4 (ref. 13) and PPR1 (ref.14).

Amino Acid Sequence↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1986

5S RNA gene specific transcription factor (TFIIIA) changes the linking number of the DNA.

The purified 5S gene specific transcription factor A (TFIIIA), when incubated with the relaxed 5S gene in the presence of topoisomerase I alters the conformation of the DNA, resulting in a change in the linking number. This interaction introduces a change of one bp per TFIIIA binding site at a low concentration of DNA to protein (1:2) which increases to an extent of 0.9 turns (9 bp) per TFIIIA binding site at a higher protein concentration (1:12). These analyses support the notion that the binding of TFIIIA to the 5S DNA introduces a minimal change in the topology of circular DNA molecules.

Animals↗
PubMed1990

Transcription factor TFIIIA stimulates DNA supercoiling promoted by a fractionated cell-free extract from Xenopus laevis.

An activity that can introduce negative supercoils into relaxed covalently closed DNA molecules has been isolated from a Xenopus laevis cell-free extract (S-150) and purified over 200-fold. The exogenous addition of ATP, other ribonucleotides and deoxyribonucleotides, as well as nonhydrolyzable analogs, stimulate DNA supercoiling which may occur by a pathway involving multiple protein components. DNA supercoiling occurs in topological single steps and is inhibited by camptothecin and berenil, but not novobiocin or VM-26, suggesting a catalytic role for topoisomerase I in the reaction. Transcription factor TFIIIA stimulates DNA supercoiling catalyzed by the isolated fraction at low factor to DNA ratios. Taken together, these results suggest that a isolated DNA supercoiling activity from the X. laevis S-150 cell-free extract can be stimulated by transcription factor TFIIIA.

Adenosine Triphosphate↗
PubMed1991

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.

Animals↗
PubMed1984

Xenopus 5S gene transcription factor, TFIIIA: characterization of a cDNA clone and measurement of RNA levels throughout development.

Initiation of 5S RNA gene transcription in Xenopus oocytes requires a 38,500 dalton polypeptide, TFIIIA. The levels of both 5S RNA and TFIIIA are regulated throughout oogenesis and embryonic development. To delineate the mechanisms by which the corresponding genes are regulated, as well as to determine the primary structure of TFIIIA, we have isolated a cDNA clone that encodes TFIIIA. Using the cDNA clone, we have determined that there is (are) one or a small number of TFIIIA gene(s) per Xenopus haploid genome, and we have estimated the size and levels of TFIIIA RNA throughout Xenopus development. We report sequence homologies between TFIIIA cDNA and regulatory regions of the Xenopus tmet and 5S RNA genes. Implications of these data for developmental regulation of the TFIIIA and 5S RNA genes are discussed.

Aging↗
PubMed1984

Altered levels of a 5 S gene-specific transcription factor (TFIIIA) during oogenesis and embryonic development of Xenopus laevis.

Xenopus laevis oocytes contain a 38,000-Da protein which serves both as a 5 S gene-specific transcription initiation factor (TFIIIA) and to stabilize 5 S RNA in ribonucleoprotein complexes. Using an antibody to this protein we have measured the levels of TFIIIA during oogenesis and embryonic development in X. laevis. The maximal steady state level (approximately 10(12) molecules/oocyte) is reached early in oogenesis but drops 10- to 20-fold in later stages and another 10- to 20-fold during ovulation. The reduced amount present in the unfertilized egg remains at a nearly constant level throughout early development, but with cell division the cellular concentration drops from 3 X 10(9) to about 10(4) molecules/cell. An immunoreactive protein of the same size is also found in liver tissues and in cultured kidney cells, which also contain about 10(4) molecules/cell. By both structural (CNBr peptide analysis) and functional (transcription of 5 S genes) analyses the embryonic and kidney cell 38,000-Da factors appear indistinguishable from oocyte TFIIIA. In addition a second antigenically related protein of about 40,000 Da is found in late stage embryos, liver tissues, and adult kidney cells (where it is severalfold more abundant than the 38,000-Da TFIIIA). The chromatographic fractionation and functional analysis of the kidney cell extracts has shown that fractions containing the 38,000-Da protein support 5 S RNA synthesis while fractions containing the 40,000-Da protein do not. The significance of these findings for 5 S gene regulation is discussed with respect to the dual function of TFIIIA, the presence of rate-limiting amounts of TFIIIA, and the possibility of stage-specific factors.

Animals↗
PubMed1988

Mapping functional regions of transcription factor TFIIIA.

Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.

Animals↗
PubMed1989

Electron microscopy reveals that transcription factor TFIIIA bends 5S DNA.

We have used a high-resolution analytical electron microscopic technique, electron spectroscopic imaging, to study the in vitro interaction between the transcription factor IIIA (TFIIIA) and 5S ribosomal gene DNA. The images and analytical measurements support our proposal that the helix axis is bent by the protein into a hairpin-shaped configuration.

Animals↗
PubMed1987

Novobiocin interferes with the binding of transcription factors TFIIIA and TFIIIC to the promoters of class III genes.

Novobiocin has been shown to inhibit class III gene transcription from both chromatin and non-chromatin templates. Since novobiocin is a well characterized inhibitor of type II DNA topoisomerases, it has been postulated that a gyrase activity is necessary for transcription. Using DNase I footprinting, we show here that novobiocin inhibits the specific binding of polymerase III transcription factors TFIIIA and TFIIIC to the promoters of the 5S RNA and VA RNA genes, respectively. Concentrations of novobiocin employed were comparable to those necessary to inhibit HeLa topoisomerase II. In vitro transcription assays, performed under equivalent conditions, demonstrated that similar novobiocin concentrations were necessary for transcription inhibition. These results strongly suggest that novobiocin interferes with transcription by inhibiting specific protein-DNA interactions.

Adenoviruses, Human↗
PubMed1988

Characterization by human antibodies of two HeLa cell proteins which are related to Xenopus laevis transcription factor TFIIIA.

The sera of two patients with autoimmune disorders recognize in HeLa cell extracts two proteins with apparent molecular masses of 37,000 (p37) daltons and 32,000 daltons (p32). These proteins are non covalently associated with 5S RNA and sediment as 7-10 S particles in sucrose density gradients. Both proteins are antigenetically related to TFIIIA, a previously described protein of Xenopus laevis, which is known as a 5S RNA transcription factor and occurs in oocytes as a noncovalent complex with 5S RNA. Like TFIIIA, HeLa cell proteins p37 binds in vitro to 5S RNA and to cloned 5S RNA genes. These results suggest that protein p37 fulfils in HeLa cells a function similar to that of TFIIIA in amphibian oocytes, ie control of 5S RNA transcription.

Animals↗
PubMed1987

[A variety of human autoantibodies recognizes in HeLa cells 2 proteins related to the TFIIIA factor of Xenopus laevis which regularizes the transcription of ribosomal 5S RNA].

Using the sera from two patients with autoimmune disorders, we have identified by immunoprecipitation of HeLa cell extracts two proteins with apparent molecular masses of 37 kDa (p 37) and 32 kDa (p 32). These proteins are associated with 5 S RNA. They are antigenetically related to Xenopus laevis 5 S RNA transcription factor TFIIIA, which is very abundant in early oocytes of this species. In contrast to what is observed in X. laevis oocytes, the TFIIIA-related proteins of HeLa cells are present in very small amounts. Our data suggest that proteins p 37 and p 32 are involved in the control of 5 S RNA transcription.

Animals↗