The primary structure of transcription factor TFIIIA has 12 consecutive repeats

Brown, Raymond S.; Sander, Christian; Argos, Patrick

doi:10.1016/0014-5793(85)80723-7

Cited by 424 publications

(225 citation statements)

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“…The UAS2 sequence includes the 25 bp directly upstream of UAS1 and allows ADR1-independent derepression of ADH2 expression (J. Yu, personal communication). ADR1 contains two zinc finger domains (10), similar to those which were first observed in transcription factor IIIA from Xenopus laevis, having the consensus sequence Cys-X24-Cys-X3-Phe-X5-Leu-X2-His-X2-His (5,19). Zinc finger domains have since been found in several eucaryotic genes thought to be involved in the regulation of transcription (6,18,25,29,30) and in some cases have been shown to bind DNA specifically at regulatory elements (8,15,28).…”

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The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2.

Eisen¹,

Taylor²,

Blumberg³

et al. 1988

Mol. Cell. Biol.

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The yeast ADR1 protein contains two zinc finger domains that are essential for its role in transcriptional activation of alcohol dehydrogenase (ADH2). These domains are thought to function as DNA-binding structures. An ADR1-,B-galactosidase fusion protein made in Escherichia coli and containing the finger domains of ADR1 binds in vitro in a zinc-dependent manner to DNA fragments containing the two ADH2 upstream activation sequences. The strongest binding is to upstream activation sequence 1, a 22-base-pair palindrome.ADR1 is a nuclear protein which is necessary for transcription of the glucose-repressible alcohol dehydrogenase gene (ADH2) in the yeast Saccharomyces cerevisiae. Repression of ADH2 is apparently maintained by posttranslational regulation of ADR1 (4, 7) rather than by DNA-binding repressors (14). The promoter of ADH2 requires upstream activation sequences (UAS) composed of two elements which function synergistically. UAS1 is a 22-base-pair (bp) inverted repeat which allows ADR1-dependent derepression of ADH2 (27). The UAS2 sequence includes the 25 bp directly upstream of UAS1 and allows ADR1-independent derepression of ADH2 expression (J. Yu, personal communication). ADR1 contains two zinc finger domains (10), similar to those which were first observed in transcription factor IIIA from Xenopus laevis, having the consensus sequence Cys-X24-Cys-X3-Phe-X5-Leu-X2-His-X2-His (5, 19). Zinc finger domains have since been found in several eucaryotic genes thought to be involved in the regulation of transcription (6,18,25,29,30) and in some cases have been shown to bind DNA specifically at regulatory elements (8,15,28). Point mutations in either finger region of ADR1 can give rise to null mutants which are incapable of activating ADH2 expression (3). To understand how ADR1 and other finger proteins bind DNA, we studied the in vitro DNAbinding properties of an ADR1-p-galactosidase fusion protein expressed in Escherichia coli.Binding of ADR1-P-galactosidase to the UAS of ADH2. A polypeptide containing amino acids 17 through 229 of ADR1 fused to P-galactosidase (H. Blumberg, Ph.D. thesis, University of Washington, Seattle, 1987), ADR1-229Z, was expressed in E. coli under the control of the strong, heatinducible A promoter, PR, on plasmid pCQ-A229Z (Fig. 1A) (23). Western blot (immunoblot) analysis of extracts with an anti-3-galactosidase antibody showed that 50% of the fusion protein was proteolyzed to B-galactosidase (data not shown). To investigate ADR1 binding to the ADH2 promoter, gel retardation analysis was performed with crude cell extracts of ADR1-229Z mixed with radiolabeled DNA fragments of the ADH2 promoter (Fig. 1B). Protein-DNA complexes of ADR1-229Z bound to radiolabeled promoter DNA are shown in Fig. 2.

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mentioning

confidence: 75%

The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2.

Eisen¹,

Taylor²,

Blumberg³

et al. 1988

Mol. Cell. Biol.

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“…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).…”

Section: Introductionmentioning

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.

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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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“…It was first recognized in the Xenopus transcription factor IIIA as a conserved protein domain of 28 to 30 amino acids that contains a number of invariant residues (3,4,41). The nuclear magnetic resonance solution structure of a single zinc finger demonstrated that this motif forms a compact globular domain that is composed of an antiparallel ␤-sheet followed by an ␣-helix and is stabilized by the coordination of a Zn 2ϩ ion through two cysteine and two histidine residues (31).…”

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confidence: 99%

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

Swirnoff

Milbrandt

1995

Molecular and Cellular Biology

299

287

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NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys 2 -His 2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T -G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently).Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins, we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.

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The primary structure of transcription factor TFIIIA has 12 consecutive repeats

Cited by 424 publications

References 18 publications

The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2.

The yeast regulatory protein ADR1 binds in a zinc-dependent manner to the upstream activating sequence of ADH2.

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

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

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