TFIIIA Binding Research Articles

Localized Frustration and Binding-Induced Conformational Change in Recognition of 5S RNA by TFIIIA Zinc Finger

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA–TFIIIA–TFIIIC– TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA–DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA.

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 Core Histone N-Terminal Tail Domains Negatively Regulate Binding of Transcription Factor IIIA to a Nucleosome Containing a 5S RNA Gene via a Novel Mechanism

Reconstitution of a DNA fragment containing a 5S RNA gene from Xenopus borealis into a nucleosome greatly restricts binding of the primary 5S transcription factor, TFIIIA. Consistent with transcription experiments using reconstituted templates, removal of the histone tail domains stimulates TFIIIA binding to the 5S nucleosome greater than 100-fold. However, we show that tail removal increases the probability of 5S DNA unwrapping from the core histone surface by only approximately fivefold. Moreover, using site-specific histone-to-DNA cross-linking, we show that TFIIIA binding neither induces nor requires nucleosome movement. Binding studies with COOH-terminal deletion mutants of TFIIIA and 5S nucleosomes reconstituted with native and tailless core histones indicate that the core histone tail domains play a direct role in restricting the binding of TFIIIA. Deletion of only the COOH-terminal transcription activation domain dramatically stimulates TFIIIA binding to the native nucleosome, while further C-terminal deletions or removal of the tail domains does not lead to further increases in TFIIIA binding. We conclude that the unmodified core histone tail domains directly negatively influence TFIIIA binding to the nucleosome in a manner that requires the C-terminal transcription activation domain of TFIIIA. Our data suggest an additional mechanism by which the core histone tail domains regulate the binding of trans-acting factors in chromatin.

Xenopus transcription factor IIIA and the 5S nucleosome: development of a useful in vitro system.

5S RNA genes in Xenopus are regulated during development via a complex interplay between assembly of repressive chromatin structures and productive transcription complexes. Interestingly, 5S genes have been found to harbor powerful nucleosome positioning elements and therefore have become an important model system for reconstitution of eukaryotic genes into nucleosomes in vitro. Moreover, the structure of the primary factor initiating transcription of 5S DNA, transcription factor IIIA, has been extensively characterized. This has allowed for numerous studies of the effect of nucleosome assembly and histone modifications on the DNA binding activity of a transcription factor in vitro. For example, linker histones bind 5S nucleosomes and repress TFIIIA binding in vitro in a similar manner to that observed in vivo. In addition, TFIIIA binding to nucleosomes assembled with 5S DNA is stimulated by acetylation or removal of the core histone tail domains. Here we review the development of the Xenopus 5S in vitro system and discuss recent results highlighting new aspects of transcription factor - nucleosome interactions,

Additivity of interactions of zinc finger motifs in specific recognition of RNA.

Xenopus transcription factor IIIA (TFIIIA) binds 5S rRNA and the 5S rRNA gene, these interactions being mediated by nine zinc fingers. To determine the contribution of each finger to the binding to 5S rRNA we prepared a series of peptides containing different numbers of zinc fingers and analyzed their interactions with RNA. The topography of these complexes was analyzed with the specific RNase and hydroxyl radical footprinting methods. Our results show the direct contribution of each zinc finger (ZF) peptide to the specific recognition of 5S rRNA. These data clearly suggest that total binding of TFIIIA with 5S rRNA is the sum of the specific interactions of the individual zinc fingers with RNA and that they have an additive character.

Contribution of individual amino acids to the nucleic acid binding activities of the Xenopus zinc finger proteins TFIIIIA and p43.

Xenopus transcription factor IIIIA (TFIIIA) binds to both 5S RNA and the 5S RNA gene in immature oocytes, an interaction mediated by nine zinc fingers. To determine the role of the central zinc fingers of the protein in these nucleic acid binding activities, a series of substitution mutants of TFIIIA were constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for DNA and RNA binding activities using a nitrocellulose filter binding assay. Finger 5, but not finger 4, 6, or 7, is involved in sequence-specific binding to the 5S RNA gene. A TWT amino acid motif in finger 6 makes a significant contribution to the binding of TFIIIA to 5S RNA, while mutations in fingers 4, 5, and 7 have little or no effect on RNA binding by TFIIIA. In striking contrast, a TWT motif in finger 6 of p43, another Xenopus zinc finger protein that binds to 5S RNA, is not necessary for 5S RNA binding by this protein. Evidence for the presence of inhibitory finger-finger interactions that limit the nucleic acid binding properties of individual zinc fingers within TFIIIA and p43 is discussed.

The role of the central zinc fingers of transcription factor IIIA in binding to 5 S RNA

In the nine-zinc finger Xenopus transcription factor TFIIIA the central group of fingers, fingers 4 to 7, have been shown to bind to 5 S RNA. In this study, we have attempted to assess the role of this region of the TFIIIA molecule in more detail than hitherto. High-resolution footprinting by RNases A and CV1 has been used to probe the binding to 5 S RNA of three TFIIIA peptides Tf(1-6), Tf(4-6) and Tf(4-7), consisting of fingers 1 to 6, 4 to 6, and 4 to 7, respectively, and of full-length TFIIIA. The results pinpoint the outer margins of binding of the central fingers within helices IV and II of TFIIIA. A comparison of the footprints reveals that the presence of finger 7 affords protection at positions C19 and U55, the distal portion of helix II and the proximal portion of loop B. In addition, our footprints suggest that the central fingers bind in the same manner, whether in an isolated group or in the intact TFIIIA molecule. In a companion study, we have determined the binding affinities of Tf(4-6) and Tf(4-7) for full-length and three truncated 5 S RNA molecules, the latter selected on the basis of the regions of the 5 S RNA molecule known to be important in the binding of TFIIIA. The analysis uses only fully active protein involved in the binding and the results are consistent with the corresponding footprinting results. This is the first time that a detailed study of the binding site of one particular zinc finger to RNA has been reported; the results should be of use in the design of 5 S RNA molecules and TFIIIA peptides for structural studies of the interaction between zinc fingers and RNA.

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.

How do linker histones mediate differential gene expression?

In developing Xenopus laevis embryos the multiple-copy oocyte-type 5S RNA genes are progressively shut down. Results presented in three recent articles 1-3 together demonstrate that replacement of the cleavage stage linker histone B4 by somatic H1 leads to chromatosomes positioned directly over these genes and adjacent sequences so as to occlude the binding site for the critical transcription factor TFIIIA. In contrast, on the somatic-type 5S genes the somatic H1 positions chromatosomes about 65 bp further upstream, thereby leaving the TFIIIA binding site exposed and the genes active. The somatic linker histone thus functions as a specific gene repressor.

TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box.

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

Structural determinants in 5S RNA and TFIIIA for 7S RNP formation.

C2H2-type zinc-finger modules define a unique structural motif, which is capable of forming specific complexes with both DNA and RNA. While the principles governing DNA binding have been defined in great detail, the mode of RNA recognition remains only poorly understood. In the absence of information from three-dimensional structural analysis of a zinc-finger/RNA complex, we have performed a number of biochemical studies to gain further insight into the molecular details of the interaction of 5S ribosomal RNA with the zinc-finger protein TFIIIA. Previous work had indicated that zinc finger 6 of TFIIIA contacts 5S RNA in close proximity or directly in the loop-A region (nucleotides 10-13). Permutation analysis of this sequence reveals that three of the four nucleotides are of vital importance for RNA recognition. Exchange of unusual and therefore characteristic aromatic residues in finger 6 against aliphatic or other aromatic amino acids reveals that the aromatic character of tryptophan 177 is essential for RNA recognition. Association with helix V in 5S RNA appears to involve specific contacts with the phosphate backbone, as evidenced by ethylation-interference assays. Introduction of multiple internal and 3'-terminal as well as 5'-terminal deletions accompanied by stabilizing sequence substitutions defines a minimal RNA fragment that is sufficient for TFIIIA binding. This RNA molecule includes a truncated/mutated helix I, helix II and helix V, as well as structurally intact loops A and E. Permutation analysis of the loop-E region emphasizes its importance for TFIIIA recognition.

Differential nucleosome positioning on Xenopus oocyte and somatic 5 s RNA genes determines both TFIIIA and H1 binding: a mechanism for selective H1 repression

In Xenopus somatic cells histone H1 effects the transcriptional repression of oocyte type 5 S RNA genes, without altering the transcription of the somatic type 5 S RNA genes. Using an unambiguous nucleosome mapping method we find substantial differences between the multiple in vitro nucleosome positions on the two types of genes. These nucleosome positions determine both transcription factor and H1 binding, allowing TFIIIA to bind more efficiently to nucleosomes containing the somatic 5 S RNA gene than to nucleosomes on the oocyte 5 S RNA gene. Significantly, in a binding competition between TFIIIA and H1, TFIIIA preferentially binds to the somatic nucleosome whereas H1 preferentially binds to the oocyte nucleosome, excluding TFIIIA binding. These results strongly suggest that nucleosome positioning plays a key role in the regulation of transcription of 5 S RNA genes and provide a molecular mechanism for the selective repression of the oocyte 5 S RNA genes by H1.

The role of zinc finger linkers in p43 and TFIIIA binding to 5S rRNA and DNA.

Transcription factor IIIA (TFIIIA) and p43 zinc finger protein form distinct complexes with 5S ribosomal RNA in Xenopus oocytes. Additionally, TFIIIA binds the internal promoter of the 5S RNA gene and supports assembly of a transcription initiation complex. Both proteins have nine tandemly repeated zinc fingers with almost identical linker lengths between corresponding fingers, yet p43 has no detectable affinity for the 5S RNA gene. TFIIIA zinc fingers 1-3 are connected by highly conserved linkers, first identified in the Drosophila protein Krüppel, that are found in many DNA binding zinc finger proteins. To understand the role of these linkers in RNA and DNA binding we exchanged three TFIIIA linker amino acids with the equivalent amino acids from p43. The major effect of linker substitution is a 50-fold reduction in DNA specificity, concomitant with an 8-fold reduction in affinity. N-Terminal zinc fingers from either TFIIIA or p43 bind to multiple specific sites on 5S RNA that are resistant to competition by tRNA or poly(rA). This mode of RNA binding is unaffected by linker substitution. These data suggest that zinc finger linkers significantly facilitate the specificity of DNA binding.

The histone binding protein nucleoplasmin does not facilitate binding of transcription factor IIIA to nucleosomal Xenopus laevis 5S rRNA genes.

In an attempt to understand the mechanism by which transcription factors compete with histone octamers for cognate binding sites in chromatin, the effect of the histone binding protein nucleoplasmin on the binding of TFIIIA to nucleosomal 5S rRNA genes was tested. In this study, it was shown that, despite the previously reported nucleosome remodeling ability of nucleoplasmin, the binding of TFIIIA to nucleosomal DNA cannot be facilitated by this protein. Furthermore, it was demonstrated that nucleoplasmin cannot overcome nucleosome mediated repression of transcription of reconstituted 5S rRNA genes. In contrast to earlier work, this study used a homologous system composed of the 5S rRNA gene, nucleoplasmin, and TFIIIA from Xenopus laevis.

Strand-specific modulation of UV photoproducts in 5S rDNA by TFIIIA binding and their effect on TFIIIA complex formation.

The relationship between UV-induced photoproduct formation and transcription factor binding was studied in a 214 bp fragment containing the entire Xenopus borealis 5S rRNA gene. DNA mobility shift and DNase I footprinting show a strong inhibition of TFIIIA binding to UV-damaged 5S rDNA. An average of approximately 2 cyclobutane pyrimidine dimers (CPDs) per 214 bp fragment, and a lesser amount of pyrimidine-pyrimidone (6-4) dimers, reduced the fraction of TFIIIA bound by approximately 70%. Furthermore, irradiation of the TFIIIA/5S rDNA complex displaces TFIIIA at doses of 0.8-2 CPDs/fragment, indicating the complex is unable to accommodate UV photoproducts. UV photofootprinting of the 50 bp TFIIIA binding region of 5S rDNA (or ICR) shows that TFIIIA binding modulates photoproduct formation primarily in the template strand. Formation of CPDs at six different sites is strongly inhibited, while another CPD site is strongly enhanced, by TFIIIA binding. Most of these sites are located in one of three boxes (A, IE, or C) designated as TFIIIA contact sites in the ICR, while one site is between these boxes. Formation of (6-4) dimers is also inhibited at several sites in the template strand by TFIIIA binding. However, formation of photoproducts in the nontemplate strand is much less affected by TFIIIA binding, where only one CPD site is inhibited in the complex. These data indicate that formation of UV photoproducts in 5S rDNA can be markedly affected by TFIIIA binding, and complex formation is inhibited by UV photoproducts.

Regulation of gene expression by small molecules.

Small molecules that target specific DNA sequences have the potential to control gene expression. Ligands designed for therapeutic application must bind any predetermined DNA sequence with high affinity and permeate living cells. Synthetic polyamides containing N-methylimidazole and N-methylpyrrole amino acids have an affinity and specificity for DNA comparable to naturally occurring DNA-binding proteins. We report here that an eight-ring polyamide targeted to a specific region of the transcription factor TFIIIA binding site interferes with 5S RNA gene expression in Xenopus kidney cells. Our results indicate that pyrrole-imidazole polyamides are cell-permeable and can inhibit the transcription of specific genes.

Protein and DNA Requirements for the Transcription Factor IIIA-induced Distortion of the 5 S rRNA Gene Promoter

Transcription factor-induced DNA distortion has become a common theme in eukaryotic gene regulation. A number of techniques have been applied to the study of transcription factor-induced DNA bending and flexibility including electron microscopy, circular permutation gel analysis, helical phasing gel analysis and cyclisation kinetics in solution. We have applied these techniques in order to assess the role that specific DNA sequences and protein domains of transcription factor IIIA (TFIIIA) play in the TFIIIA-induced distortion of the Xenopus5 S ribosomal RNA gene promoter. Electron spectroscopic imaging analysis of TFIIIA : DNA com- plexes indicate that TFIIIA binding involves compaction of the 5 S promoter into a precise three-dimensional hairpin-shaped structure. This compaction can be detected utilising circular permutation gel analysis and the distortion results in an apparent bend angle of 55 to 60° near the centre of the TFIIIA binding site. Helical phasing analysis demonstrates that the 60° bend angle as measured by circular permutation can be detected as a static bend directed towards the minor groove between bases +63 and +64 of the 5 S rRNA gene. The amplitude of the TFIIIA : 5 S gene phasing signal is similar to the phasing signal obtained utilising bacterial CAP : DNA complexes with bend angles of approximately 90°. These results are supported by phased ligase-mediated cyclisation kinetics in solution. Analysis of DNA deletion constructs indicate that the 5′ A block of the internal 5 S gene promoter, which is required for transcriptional activity, is also required for TFIIIA-induced distortion of the 5 S gene promoter. Analysis of the N-terminal papain fragment of TFIIIA indicates that the 34 kDa zinc finger DNA binding domain is sufficient for compaction of the 5 S gene promoter. These results are discussed in relation to the modular model of TFIIIA : DNA interaction in which individual zinc fingers contribute to the protein-induced distortion of the DNA helix and overall DNA binding affinity in a complex, non-additive fashion.

Analysis of the Binding of Xenopus Transcription Factor IIIA to Oocyte 5 S rRNA and to the 5 S rRNA Gene

Binding of transcription factor IIIA (TFIIIA) to site-specific mutants of Xenopus oocyte 5 S rRNA has been used to identify important recognition elements in the molecule. The putative base triple G75:U76:A100 appears to determine the conformation of the loop E region whose integrity is especially important for binding of the factor. Proximal substitutions in helices IV and V indicate that the proper folding of loop E is also dependent on these structures. Mutations in helix V affect binding of TFIIIA to 5 S rRNA and to the gene similarly and provide evidence that zinc finger 5 makes sequence-specific contact through the major groove of both nucleic acids. Although fingers 1-3 are positioned along helix IV and loop D, mutations in this region, including those that disrupt the tetraloop or close the opening in the major groove of the helix created by the U80:U96 mismatch, have no impact on binding. Substitutions made at stem-loop junctions in the arm of the RNA comprised of helix II-loop B-helix III display minor decreases in affinity for TFIIIA. Despite the alignment of the factor along nearly the entire length of 5 S rRNA, the essential elements for high affinity binding are limited to the central region of the molecule. Analysis of the corresponding mutations in the gene confirm that box C and the intermediate element provide the high affinity sites for binding of the factor to the DNA. Despite the small thermodynamic contribution made by contacts to box A, mutations made in this element can cause substantial changes in the orientation of the carboxyl-terminal fingers along the 5'-end of the internal control region.

Human TFIIIA alone is sufficient to prevent nucleosomal repression of a homologous 5S gene.

Plasmid DNA harbouring the human 5S rRNA gene was assembled into nucleosomes using either Xenopus S150 extracts or purified core histones in the presence of pectin. In both cases reconstitution of nucleosomes led to a complete repression of transcription. This repression could be efficiently counteracted by preincubating the template DNA with highly purified hTFIIIA which allowed the protein to bind to the ICR of the 5S gene. By using an efficient and well-defined in vitro reconstitution system based on isolated core histones in the presence of pectin, which is devoid of endogenous transcription factors, we demonstrate here for the first time that human TFIIIA alone is sufficient to prevent nucleosomal repression of h5S gene transcription and that additional pol III transcription factors are not required to achieve this effect. Additionally, we investigated the binding of hTFIIIA to a mononucleosome reconstituted on the human 5S gene. DNAse I footprinting experiments reveal that the entire ICR of the human 5S gene is covered by the nucleosome, thereby precluding the subsequent binding of human TFIIIA to the promoter of the 5S gene.