Contribution of Individual Base Pairs to the Interaction of TFIIIA with the Xenopus 5S RNA Gene

Veldhoen, Nik; You, Qimin; Setzer, David R.; Romaniuk, Paul J.

doi:10.1021/bi00190a009

Cited by 34 publications

(45 citation statements)

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“…Prior methylation of certain guanine bases or ethylation of several phosphates interferes with the binding of TFIIIA (10,34). 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.…”

Section: Resultsmentioning

confidence: 99%

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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.

233

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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Section: Resultsmentioning

confidence: 99%

“…It has been proposed, based on results from DNA methylation protection and binding interference and on site-directed mutagenesis experiments (8,(33)(34)(35)(36)(37), that these fingers wrap, like fingers 1-2-3, around the major groove of base pairs ϩ48 to ϩ62 (9, 10). In the model shown in Fig.…”

Section: Discussionmentioning

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.

233

185

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“…The purification of recombinant TFIIIA from Escherichia coli cells harboring the expression plasmid pTF3 was carried out as described previously (44). The technique of Ausió et al (2) was used for the isolation of nucleosome core particles from chicken erythrocytes.…”

Section: Methodsmentioning

confidence: 99%

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

Howe

Ausió

1998

Molecular and Cellular Biology

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We sought to study the binding constraints placed on the nine-zinc-finger protein transcription factor IIIA (TFIIIA) by a histone octamer. To this end, five overlapping fragments of the Xenopus laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes, and it was subsequently shown that nucleosome translational positioning is a major determinant of the binding of TFIIIA to the 5S rRNA genes. Furthermore, it was found that histone acetylation cannot override the TFIIIA binding constraints imposed by unfavorable translational positions.Xenopus laevis produces two major types of 5S rRNA: the somatic type is synthesized in most cell types, whereas the oocyte type is synthesized during early oogenesis, during embryogenesis, and in certain tissue culture cell lines (14,47). Each 5S rRNA type is transcribed from a distinct multigene family, and considerable research has focused on understanding the differential expression of these genes. One hypothesis suggests that the transcription complexes which form on the oocyte genes are relatively unstable compared to those of the somatic counterparts. As a result of this, the oocyte genes are transcribed only when transcription factor IIIA (TFIIIA) levels are relatively high, as is the case during oogenesis. In somatic cells, in which TFIIIA levels are much lower, the transcription complexes dissociate from the oocyte gene, allowing the subsequent assembly of repressive nucleosome structures which preclude further factor binding (47). Conflicting with this hypothesis are results which show that, at least in vitro, the RNA polymerase III transcription complexes, once formed, have similar stabilities on both the oocyte and somatic genes (35). Thus, at this time, the reasons for the differential transcription of these two gene families in X. laevis are not fully understood.The results of 5S rRNA gene transcription studies, performed with both native and reconstituted chromatin as templates, have been conflicting. In one such study it was demonstrated that chromatin isolated from a Xenopus kidney cell line can serve as a template for transcription of the oocyte gene after the removal of histone H1 (34). In addition, it has been shown that incorporation of a somatic histone H1 variant into chromatin during embryogenesis results in specific repression of TFIIIA-activated oocyte 5S rRNA transcription (8). Furthermore, histone H1 has been shown to specifically repress transcription of oocyte genes in reconstituted chromatin while leaving the corresponding somatic genes unaffected (40). These results suggest that it is the presence of histone H1 which is responsible for the repression of oocyte transcription. This is not surprising considering that histone H1 is thought of as a repressor of transcription, although the exact mechanism of this repression is not known. In contrast to the abovementioned work, studies with chromatin reconstituted in the absence of linker histones have shown that the presence of nucleosome core particles alone is sufficient to repress oocyt...

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“…This collection includes all possible single point mutations between and including bp 45 and 96 in the 5 S rRNA gene; a number of the mutants have been analyzed previously with respect to TFIIIA binding affinity (22). In the current study, labeled DNA fragments containing the wild-type or mutant gene were prepared from these plasmids using the PCR primer 759 (5Ј-CGGGGAAAGAAAAATGCATG-3Ј) and either primer 190 (5Ј-GCTTGCATGCCACGCGTCTC-3Ј) or primer 290 (5Ј-GCCTACGGC-CATACCACCCT-3Ј).…”

Section: Methodsmentioning

confidence: 99%

Is There a Dynamic DNA-Protein Interface in the Transcription Factor IIIA-5 S rRNA Gene Complex?

Brady

Setzer

2005

Journal of Biological Chemistry

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) have proposed that several amino acid side chains exhibit considerable conformational mobility at the DNA-protein interface in the transcription factor IIIA⅐5 S rRNA gene complex and that the rapid movements of these side chains permit them to make fluctuating contacts with adjacent bp in the DNA target site. This "dynamic interface" model makes biochemical predictions concerning the consequences of truncating specific amino acid side chains and the effects of these truncations on sequence selectivity in DNA binding. The model also makes predictions concerning the effects of DNA sequence context on the apparent energetic contributions to binding made by individual bp. We have tested these predictions, and our results are inconsistent with any significant energetic role being played by the contact of multiple bp by conformationally mobile amino acid side chains. They do, however, show that some individual amino acids affect the recognition of multiple bp through mechanisms other than direct interaction.Transcription factor IIIA (TFIIIA) 1 from Xenopus laevis exhibits a number of unusual features that make it of special interest in the study of nucleic acid-protein interactions. These include its ability to recognize both DNA (the internal control region of the 5 S rRNA gene) and RNA (5 S rRNA) with high affinity and specificity (1-4), its unusually large number of zinc fingers (nine), and correspondingly large DNA binding site (5-8), the complex thermodynamics with which it recognizes both DNA (9, 10) and RNA (11, 12), and its status as the archetypal zinc finger protein (13). Although it has been the subject of numerous biochemical studies, direct determination of the structure of the TFIIIA⅐5 S rRNA gene complex has been limited to two TFIIIA fragments bound to portions of the 5 S rRNA gene internal control region. Wright and colleagues (14, 15) have used nuclear magnetic resonance methods to determine the structure of the first three zinc fingers of TFIIIA bound to a 13-bp fragment of the 5 S rRNA gene, and Nolte et al. (16) have described a structure for the first six zinc fingers of TFIIIA bound to a 32-bp fragment of the same gene in a DNA-protein co-crystal. The structural models resulting from these studies confirmed the existence of some DNA-protein interactions in the TFIIIA⅐5 S rRNA gene complex that were predicted from the previously determined structures of other zinc finger proteins of the TFIIIA class (17-20) and also revealed previously undescribed features of DNA recognition by a zinc finger protein.Among the most interesting novel features of recognition proposed by Wright and colleagues was the existence of a dynamic DNA-protein interface in the complex of the three N-terminal zinc fingers of TFIIIA bound to bp 80 -92 in the 5 S rRNA gene. In particular, Foster et al. (15) and Wuttke et al. (14) proposed that several amino acid side chains involved in direct contact with DNA bp in the complex undergo rapid conformational fluctuations (in the s-ms time range). Furthermore, the authors propo...

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Contribution of Individual Base Pairs to the Interaction of TFIIIA with the Xenopus 5S RNA Gene

Cited by 34 publications

References 55 publications

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

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

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

Is There a Dynamic DNA-Protein Interface in the Transcription Factor IIIA-5 S rRNA Gene Complex?

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