Protein Transcription Factor IIIA Research Articles

Ribosomal protein L5 and transcription factor IIIA from Arabidopsis thaliana bind in vitro specifically Potato spindle tuber viroid RNA

Potato spindle tuber viroid (PSTVd) contains an element of tertiary structure -loop E- also present in eukaryotic 5S rRNA. The ribosomal protein L5 and transcription factor IIIA (TFIIIA) from Arabidopsis thaliana bind 5S rRNA in vitro and in vivo, mediating different functions that include nucleocytoplasmic transport and transcription activation, respectively. We show that A. thaliana L5 and TFIIIA also bind PSTVd (+) RNA in vitro with the same affinity as they bind 5S rRNA, whereas the affinity for a chloroplastic viroid is significantly lower. These two proteins might participate in the synthesis and delivery of PSTVd RNA in vivo.

The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation

An account is given of the discovery of the classical Cys(2)His(2) zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of structural studies on its structure and its interaction with DNA. The finger is a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome. The zinc finger design is ideally suited for engineering proteins to target specific genes. In the first example of their application in 1994, a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In addition, a reporter gene was activated by targeting an inserted zinc finger promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that by combining zinc fingers with other effector domains, e.g., from nucleases or integrases, to form chimeric proteins, genomes could be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of therapeutic importance.

The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation

A long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically. Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome. The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated. The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified. Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.

DNA repair in a protein–DNA complex: searching for the key to get in

An obstacle encountered by nucleotide excision repair (NER) proteins during repair of the genome is the masking of bulky lesions by DNA binding proteins. For example, certain transcription factors are known to be impediments, and suppress damage removal at their recognition sequences. We have used well-defined protein–DNA complexes to study the molecular mechanism(s) used by repair proteins in gaining access to DNA lesions in chromatin. Using transcription factor IIIA (TFIIIA) and the 5S ribosomal RNA gene (5S rDNA), we previously measured position-dependent effects of cyclobutane pyrimidine dimers (CPDs) at five different sites within the internal control region (ICR) on complex formation [Y. Kwon, M.J. Smerdon, Binding of zinc finger protein transcription factor IIIA to its cognate DNA sequence with single UV photoproducts at specific sites and its effect on DNA repair, J. Biol. Chem. 278 (2003) 45451–45459]. We found that CPDs at two of these sites enhance the TFIIIA–rDNA dissociation rate, which correlates with enhanced repair at these two sites. Here, we used a novel approach to directly compare dissociation of randomly damaged rDNA with NER. We refined the relationship between dissociation and repair of the complex by examining all CPD sites in the transcribed strand. A 214 bp 5S rDNA fragment was irradiated with UV light to produce CPDs at dipyrimidine sites and ∼1 CPD per fragment. Positions of CPDs that alter binding of TFIIIA were determined by T4 endonuclease V mapping of TFIIIA-bound and unbound fractions of UV-irradiated DNA. As expected, the results reveal that dissociation of TFIIIA from the complex is significantly enhanced by CPDs within the ICR. Moreover, the levels of dissociation induced by CPDs were quantitatively compared with their repair efficiency, and indicate that repair rates of most CPDs in the complex closely correlate with the dissociation rates. In addition, changes in dissociation rate are similar to changes in CPD frequency induced by TFIIIA binding. These findings indicate that structural compatibility of a DNA lesion within a protein–DNA complex can determine both lesion frequency and repair efficiency.

The discovery of zinc fingers and their development for practical applications in gene regulation

An account is given of the discovery in 1985 of the classical Cys 2 His 2 (C 2 H 2 ) zinc finger, aris- ing from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of subsequent structural studies on its 3D structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner structural hydrophobic core. This work showed not only a new pro- tein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of different lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that this zinc finger is found widespread in nature, in 3% of the genes of the human genome. It had long been the goal of molecular biologists to design DNA binding proteins for control of gene expres- sion and we have adopted the zinc finger design and principle for this purpose. We demonstrated that the zinc finger design is ideally suited for such purposes, discriminating between closely related DNA sequences both in vitro and in vivo, and we have therefore adapted this natural design for engineering zinc finger proteins for targeting specific genes. The first example of the potential of the method was published in 1994 when a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In the same paper we also showed that we could activate a reporter gene by target- ing a nine base pair promoter which we had inserted. Thus by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. By combining the targeting zinc fingers with other effector or functional domains e.g. from nucleases or integrases, to form chimeric proteins, genomes can be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of potential therapeutic importance.

Binding of Zinc Finger Protein Transcription Factor IIIA to Its Cognate DNA Sequence with Single UV Photoproducts at Specific Sites and Its Effect on DNA Repair

The relationship between DNA repair efficiency at specific locations in the binding site of the nine-zinc finger protein transcription factor IIIA (TFIIIA) and binding of its individual zinc fingers was studied. Homogeneously damaged oligonucleotides, which contained a single cis-syn cyclobutane thymine dimer (CTD) at one of six different sites in the internal control region (ICR) of the 5 S rRNA gene to generate a series of damaged DNA substrates, were prepared by chemical synthesis. Binding of TFIIIA to the substrates was assayed by measurement of dissociation constants (Kd), dissociation rates (koff), and protein-DNA contacts. The results indicated that a single CTD in the ICR does not significantly affect the Kd of TFIIIA. In contrast, CTDs at positions +55 and +72 (from the transcription start site) in the ICR markedly enhanced koff of TFIIIA from the complex. In addition, CTDs in these two sites increased methylation of the N7 of guanines (by dimethyl sulfate) in the zinc finger contacts of the ICR-TFIIIA complex. Furthermore CTDs at +55 and +72 were more efficiently removed from the complex than CTDs at other sites in the ICR by Xenopus oocyte nuclear extracts. This suggests that repair of CTDs closely correlates with changes in the binding of individual zinc fingers of the ICR-TFIIIA complex. These results have implications for the mechanism of DNA damage recognition and repair in protein-DNA complexes.

Influence of TFIIIA-type linker at the N- or C-terminal of nine-zinc finger protein on DNA-binding site

The central three-zinc finger connection of the native nine-zinc finger protein transcription factor IIIA (TFIIIA) is composed of unique linker sequences, –NIKICV–, –TQQLP–, –AG–, and –QDL–. New artificial nine-zinc finger proteins, Sp1ZF9TC and Sp1ZF9TN, which use the TFIIIA-type linker for their C- and N-terminal three-zinc finger connections have been created. To investigate the influence of TFIIIA-type linker sequences by their different locations in the proteins, gel mobility shift assays (GMSA), DNase I footprinting assays, methylation interference analyses, and hydroxyl radical footprinting assays were performed. The GMSA revealed similar DNA-binding affinities of these two proteins. The footprinting analyses indicated that the two zinc finger proteins recognize the same part of GCII or GCIII DNA. Moreover, the specific base contacts were observed in the same sites of the substrate DNA. In the present proteins, Sp1ZF9TC and Sp1ZF9TN, the four zinc fingers (fingers 1–4 or 5–9) situated in the site opposite to the TFIIIA-type linker position participate in their DNA bindings. The position of the TFIIIA-type linker is important in DNA recognition by multi-zinc finger proteins.

Repetitive zinc-binding domains in the protein transcription factor IIIA fromXenopus oocytes

Repetitive zinc-binding domains in the protein transcription factor IIIA fromXenopus oocytes

Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

Nucleosome translational position, not histone acetylation, determines TFIIIA binding to nucleosomal Xenopus laevis 5S rRNA genes.

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.

Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus transcription factor IIIA

Zinc finger proteins of the Cys2His2 class are DNA sequence-specific transcription factors. Previous structural studies of zinc finger protein-DNA complexes have shown that amino acids in the finger tip and alpha-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein transcription factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that a polypeptide containing zinc fingers 1-5 (zf1-5) binds oligonucleotides with modifications in the major groove of the finger 4 binding site with wild-type affinity. Mutagenesis and binding site selection studies were performed to determine whether high affinity DNA binding by zf1-5 requires a particular sequence in the binding site for finger 4. Several mutations in this region of the 5S gene reduced the DNA-binding affinity of zf1-5; however, selection and amplification binding assays did not recover the wild-type finger 4 binding site sequence from a pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on the top strand was highly selected within the finger 4 binding site. We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure rather than by a unique DNA sequence. Deletion of finger 4 from zf1-5 results in a protein with poor binding affinity, demonstrating the importance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions between fingers.

Zinc induces a bend within the transcription factor IIIA-binding region of the 5 S RNA gene

Binding of Zn 2+ to the 5 S RNA gene sequence of Xenopus borealis results in strong bending of the DNA, as inferred from transient electric birefringence data. The effect is specific for Zn 2+; several other divalent ions are not able to induce a bend of a similar magnitude. Using five different fragments that span the binding sequence, we are able to estimate a bend magnitude of at least 55 ° centered at base-pair +65 within the gene. This places the bend within the binding domain of the gene-regulatory protein transcription factor (TF) IIIA. Recent evidence has shown that the protein-DNA complex is also bent. Although our data do not allow us directly to link the two bends, our results suggest that TFIIIA could form a folded structure by stabilizing the same bent conformation that is induced by binding of Zn 2+. The chemistry of Zn 2+ binding to DNA, and the sequence around the bend center, suggest that the bend is most probably caused by joint co-ordination of Zn 2+ to the N-7 groups of stacked purine residues.

Single crystals of long DNA molecules.

We have grown single crystals of long DNA molecules, 54 and 65 base pairs in length which encompass the binding site of the Xenopus protein transcription factor IIIA (TFIIIA). These molecules are considerably longer than those previously crystallised. X-ray diffraction shows that the crystals are ordered to a resolution of 9 A. The DNA molecules are arranged side to side in layers which are orientated at 120 degrees with respect to adjacent layers. The phosphate backbones of molecules in adjacent layers are interdigitated in both the major and minor grooves. The intensity distribution indicates that the average structure is B-like, although CD spectra in solution are more consistent with the structure, intermediate between A- and B-, found in crystals of a nonamer fragment of the binding site. The B-character in our crystals may therefore be impressed by the tight packing between layers.

A novel method for the purification of the Xenopus transcription factor IIIA

We have developed a quick and simple purification method that yields large quantities of the Xenopus zinc finger protein transcription factor IIIA (TFIIIA). The protein is purified in the form of the 7S storage particle (TFIIIA/5S RNA complex) found in the ovaries of immature Xenopus. Our method yields 0.5 to 1 mg of pure 7S particle per ovary. It involves a high speed centrifugation step, fractionation on a gel filtration column and a precipitation step using calcium chloride. TFIIIA purified using this protocol retains full DNA binding activity and has the expected zinc ion content.

EXAFS study of the zinc-binding sites in the protein transcription factor IIIA.

The protein transcription factor IIIA (TFIIIA) is involved in the synthesis of 5S RNA in vitro by RNA polymerase III. It can be isolated from Xenopus laevis oocytes as a 7S particle in which the protein is associated with 5S RNA. Recently it has been shown that the native particle contains 7-11 zinc atoms. Analysis of the amino-acid sequence of TFIIIA revealed nine similar domains of approximately 30 amino acids, each containing two invariant pairs of histidines and cysteines, which have been implicated as possible binding sites for the zinc atoms. Other regulatory proteins with sequence homology to the zinc-binding domains of TFIIIA have now been reported. Here, we report the results of an EXAFS (extended X-ray absorption fine structure) study of TFIIIA which shows that the coordination sphere of the zinc sites consists of two cysteine and two histidine residues.

Repetitive Zn2+-binding domains in the protein transcription factor IIIA from Xenopus oocytes

Repetitive Zn2+-binding domains in the protein transcription factor IIIA from <i>Xenopus</i> oocytes

Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes.

The 7S particle of Xenopus laevis oocytes contains 5S RNA and a 40-K protein which is required for 5S RNA transcription in vitro. Proteolytic digestion of the protein in the particle yields periodic intermediates spaced at 3-K intervals and a limit digest containing 3-K fragments. The native particle is shown to contain 7-11 zinc atoms. These data suggest that the protein contains repetitive zinc-binding domains. Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc. The linear arrangement of these repeated, independently folding domains, each centred on a zinc ion, comprises the major part of the protein. Such a structure explains how this small protein can bind to the long internal control region of the 5S RNA gene, and stay bound during the passage of an RNA polymerase molecule.