Relative Contributions of the Zinc Fingers of Transcription Factor IIIA to the Energetics of DNA Binding

Clemens, Karen R.; Zhang, Penghua; Liao, Xiubei; McBryant, Steven J.; Wright, Peter E.; Gottesfeld, Joel M.

doi:10.1006/jmbi.1994.1701

Cited by 65 publications

(75 citation statements)

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“…Although groups of Zn-fingers from TFIIIA, e.g. 1-3 through 1-6, have similar binding constants for their cognate DNA, on the order of 10 9 , we were unable to demonstrate binding of Zn n -TFIIIA to the ICR with less than n = 7 ± 2 [51]. Zn-TFIIIA with different Zn 2+ stoichiometries displayed no significant differences in its kinetic lability or thermodynamic stability in the various reactions carried out to characterize the metal binding sites (Tables 2 and 3).…”

Section: Discussionmentioning

confidence: 63%

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

Huang

Krepkiy

et al. 2004

Journal of Inorganic Biochemistry

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Properties of the metal ion binding sites of Zn-transcription factor IIIA (TFIIIA) were investigated to understand the potential of this type of zinc finger to undergo reactions that remove Zn 2+ from the protein. Zn-TFIIIA was purified from E. coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn 2+ was shown to depend on the details of the isolation process. The average dissociation constant of Zn 2+ in Zn-TFIIIIA was 10 −7 . The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0 × 10 −8 . The reactivity of Zn-TFIIIA with a series of metal binding ligands, including 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene (zincon), 4-(2-pyridylazo)-resorcinol (PAR), and 3-ethoxy-2-oxo-butyraldehyde-bis-(N 4 -dimethylthiosemicarbazone) (H 2 KTSM 2 ) revealed similar kinetics. The reactivity of PAR with Zn-TFIIIA declined substantially when the protein was bound to the internal control region (ICR) of the 5S ribosomal DNA. Both Cd 2+ and Pb 2+ disrupt TFIIIA binding to its cognate DNA sequence. The Pb 2+ dissociation constant of Pb-F3 was measured as 2.5 × 10 −8 . According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb 2+ .

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Section: Discussionmentioning

confidence: 63%

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

Huang

Krepkiy

et al. 2004

Journal of Inorganic Biochemistry

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“…Serine-16, the primary site of phosphorylation, is located within the ␤-turn of the first zinc finger of TFIIIA. There is considerable evidence that much of the free energy of binding of TFIIIA to the 5S rRNA genes is derived from the first three zinc fingers (11,12,15,17,43,48,69). Thus, it seemed reasonable that phosphorylation or mutations at serine-16 might affect the binding of TFIIIA to either or both 5S rRNA genes.…”

Section: Resultsmentioning

confidence: 99%

Restricted Specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes

Ghose

Malik

Huber

2004

Molecular and Cellular Biology

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Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA. The wild-type factor and the alanine variant activate transcription of somatic-and oocyte-type 5S rRNA genes in nuclear extract immunodepleted of endogenous TFIIIA. The glutamic acid variant (S16E) supports the transcription of somatic-type genes at levels comparable to those of wild-type TFIIIA; however, there is no transcription of the oocyte-type genes. This differential behavior of the phosphomimetic mutant protein is also observed in vivo when using early-stage embryos, where this mutant failed to activate transcription of the endogenous oocyte-type genes. Template exclusion assays establish that the S16E mutant binds to the oocyte-type 5S rRNA genes and recruits at least one other polymerase III transcription factor into an inactive complex. Phosphorylation of TFIIIA by CK2 may allow the factor to continue to act as a positive activator of the somatic-type genes and simultaneously as a repressor of the oocyte-type 5S rRNA genes, indicating that there is a mechanism that actively promotes repression of the oocyte-type genes at the end of oogenesis.The synthesis of 5S rRNA during Xenopus oogenesis and embryogenesis provides a paradigm for developmental control of transcription (79). The oocyte-type 5S rRNA genes, which number more than 20,000 per haploid genome, are transcribed during oogenesis and briefly during early embryogenesis. The 400 somatic-type genes are active at all stages of development. Formation of transcription initiation complexes on the internal promoters of the 5S rRNA genes requires the initial binding of transcription factor IIIA (TFIIIA), followed by the ordered addition of TFIIIC and TFIIIB (46). Despite minor differences in the sequences of the two types of 5S rRNA genes, TFIIIA binds to the internal promoters of both with equal affinity (52). TFIIIC, however, preferentially binds to and stabilizes the complex of TFIIIA on the somatic-type genes (44, 79). Thus, the differential transcription of the two 5S rRNA genes during early development could, at least in part, be due to the levels of transcription factors. Nonetheless, the principal mediator of 5S rRNA gene transcription from gastrulation onward is chromatin structure.Histone H1 orchestrates the repression of oocyte-type genes in somatic cells (5,21,66,78). This inhibition occurs after the midblastula transition (MBT), when adult H1A begins to replace the maternal histone H1 variant, H1M (20,21,40). Nucleosomes are arranged differently over the two types of 5S RNA genes in somatic cells (10, 30, 83) because histone H1 acts as an architectural determinant (58, 67). In the presence of the linker histone, a stable nucleosome is positioned on the oocyte 5S rRNA gene that prevents binding of TFIIIA,...

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“…Site-directed mutagenesis of base pairs within the ICR decreases transcriptional activity (8,35) or lowers TFIIIA binding (36,37). Some amino acid mutations in TFIIIA linkers (38)(39)(40) or zinc fingers (41) decrease TFIIIA binding to the ICR. A recent summary of biochemical and genetic studies on fingers 1-2-3 may be found in ref.…”

Section: Resultsmentioning

confidence: 99%

Differing roles for zinc fingers in DNA recognition: Structure of a six-finger transcription factor IIIA complex

Nolte

Conlin

Harrison

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

230

185

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The crystal structure of the six NH 2 -terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound with 31 bp of the 5S rRNA gene promoter has been determined at 3.1 Å resolution. Individual zinc fingers are positioned differently in the major groove and across the minor groove of DNA to span the entire length of the duplex. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.TFIIIA is an essential component of the RNA polymerase III (Pol III) transcription initiation complex for 5S rRNA in Xenopus laevis oocytes (1-3). TFIIIA also participates in the nuclear export (4) and storage of 5S rRNA, with which it forms a stable cytoplasmic 7S particle (5). The DNA-binding site for a single TFIIIA protein extends over 55 bp of the 5S rRNA gene promoter (6, 7). This site lies within the 5S rRNA coding sequence itself. It is effectively a tripartite promoter (8) containing separated ''box A,'' ''intermediate element'' (IE), and ''box C'' sequences ( Fig. 1 A). Similar regulatory elements exist in tRNA gene promoters. Mapping the details of this extensive protein-DNA interaction using chemical, biochemical, and genetic techniques has continued for almost 20 years (1-3, 9, 10). The discovery of nine zinc fingers in TFIIIA (11,12) led to the notion of a transcription factor with repeated modules in its DNA-binding domain (Fig. 1B).Our present knowledge of how zinc fingers bind specifically to DNA comes largely from several x-ray structures (13)(14)(15)(16)(17)(18). In all of these protein-DNA complexes, there are contiguous zinc finger interactions with base pairs in the major groove. In Zif268, for example, three fingers recognize successive, overlapping base pair quartets in the major groove, covering a total of 10 bp. In the DNA complex of a five-finger segment from Gli, the first finger lies outside the major groove and makes no DNA contacts. The remaining fingers wrap in the major groove rather like those of Zif268. An extension of this mode of binding is not sufficient to explain the size of the TFIIIA-binding site, however (see, for example, models proposed in refs. 9 and 10). The NH 2 -terminal six zinc fingers of TFIIIA bound in a complex with 31 bp of the 5S rRNA gene has been reconstituted (19) and crystallized. We report here the structure of the complex at 3.1 Å resolution (Table 1) and describe how its zinc fingers interact with DNA in different ways. An NMR structure of fingers 1-2-3 bound to DNA has recently been published (20, 21). MATERIALS AND METHODSProtein and DNA Oligonucleotides. Recombinant TFIIIA (amino acid residues 1-190) was produced from plasmid pRSET B (Invitrogen) in Escherichia coli BL21(DE3). After sonication the protein was extracted in 7 M urea from cell pellets and purified on Bio-Rex 70 (Bio-Rad) and heparin Sepharose columns. Synthetic oligonucleotides were purified by MonoQ (Pharmacia) chromatography in 7 M urea. Thymines were replaced by 5-iododeoxyurac...

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Relative Contributions of the Zinc Fingers of Transcription Factor IIIA to the Energetics of DNA Binding

Cited by 65 publications

References 0 publications

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

Restricted Specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes

Differing roles for zinc fingers in DNA recognition: Structure of a six-finger transcription factor IIIA complex

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