Transcription Factor TFIIIA Research Articles

Malaria parasites possess a telomere repeat-binding protein that shares ancestry with transcription factor IIIA.

Telomere repeat-binding factors (TRFs) are essential components of the molecular machinery that regulates telomere function. TRFs are widely conserved across eukaryotes and bind duplex telomere repeats via a characteristic MYB-type domain. Here, we identified the telomere repeat-binding protein PfTRZ in the malaria parasite Plasmodium falciparum, a member of the Alveolate phylum for which TRFs have not been described so far. PfTRZ lacks an MYB domain and binds telomere repeats via a C2H2-type zinc finger domain instead. In vivo, PfTRZ binds with high specificity to the telomeric tract and to interstitial telomere repeats upstream of subtelomeric virulence genes. Conditional depletion experiments revealed that PfTRZ regulates telomere length homeostasis and is required for efficient cell cycle progression. Intriguingly, we found that PfTRZ also binds to and regulates the expression of 5S rDNA genes. Combined with detailed phylogenetic analyses, our findings identified PfTRZ as a remote functional homologue of the basic transcription factor TFIIIA, which acquired a new function in telomere maintenance early in the apicomplexan lineage. Our work sheds unexpected new light on the evolution of telomere repeat-binding proteins and paves the way for dissecting the presumably divergent mechanisms regulating telomere functionality in one of the most deadly human pathogens.

Visualizing protein interactions involved in the formation of the 42S RNP storage particle of Xenopus oocytes

During early phases of Xenopus oogenesis, 5S rRNA and tRNAs are stored in the cytoplasm of young oocytes in the form of a common RNA-protein complex termed the 42S particle. These storage particles comprise two kinds of proteins with different RNA binding specificities. The tRNA-binding protein 42Sp50 belongs to the EF1A (eukaryotic translation elongation factor 1A) family of translation elongation factors, while 42Sp43 is a diverged form of the transcription factor TFIIIA (transcription factor IIIA) and binds 5S rRNA. Little is known about the mode of protein-protein interactions that stabilize the 42S particle. We have determined the intracellular localization of the protein components of the 42S particle by expressing fluorescent protein-tagged fusions in transparent previtellogenic oocytes. 42Sp50 and its isoforms (EF1A-S and EF1A-O) were excluded from the nuclei and distributed uniformly throughout the cytoplasm with no enrichment in the Balbiani bodies, as described earlier by immunocytochemistry. In contrast, 42Sp43 accumulated in the amplified nucleoli. However, when both proteins were simultaneously expressed, 42Sp43 was no longer present in the nucleoli but was retained, together with 42Sp50, in the cytoplasm, the most likely site of 42S particle assembly. In contrast, the somatic-type EF1A isoforms were unable to redirect 42Sp43 from the nucleolar to the cytoplasmic compartment. We also tested for in vivo interactions using transiently transfected mammalian cells (COS-7 cell line). In this heterologous cell system 42Sp43 remained bound to the nucleoli but, on co-expression, induced the redistribution of 42Sp50 from the cytoplasm to the nucleoli. The microscopic approach described allows visualization of protein-protein interactions involved in the assembly of 42S storage particles. In particular, the transfection assay using COS-7 cells provides a rapid screening test that should facilitate identification of critical residues and structural determinants that enable the proteins of the 42S storage particle to interact with each other and to establish distinct higher-order RNP (ribonucleoprotein) complexes.

Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA

Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

Crystal structure of a zinc-finger-RNA complex reveals two modes of molecular recognition.

Zinc-finger proteins of the classical Cys2His2 type are the most frequently used class of transcription factor and account for about 3% of genes in the human genome. The zinc-finger motif was discovered during biochemical studies on the transcription factor TFIIIA, which regulates the 5S ribosomal RNA genes of Xenopus laevis. Zinc-fingers mostly interact with DNA, but TFIIIA binds not only specifically to the promoter DNA, but also to 5S RNA itself. Increasing evidence indicates that zinc-fingers are more widely used to recognize RNA. There have been numerous structural studies on DNA binding, but none on RNA binding by zinc-finger proteins. Here we report the crystal structure of a three-finger complex with 61 bases of RNA, derived from the central regions of the complete nine-finger TFIIIA-5S RNA complex. The structure reveals two modes of zinc-finger binding, both of which differ from that in common use for DNA: first, the zinc-fingers interact with the backbone of a double helix; and second, the zinc-fingers specifically recognize individual bases positioned for access in otherwise intricately folded 'loop' regions of the RNA.

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.

Energetics of the specific binding interaction of the first three zinc fingers of the transcription factor TFIIIA with its cognate DNA sequence.

The energetics of the specific interaction of a protein fragment (zf1-3) containing the three N-terminal zinc fingers of the Xenopus laevis transcription factor TFIIIA with its cognate DNA sequence, contained in a 15 bp DNA duplex were studied using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and fluorescence titration. The use of both ITC and DSC is necessary to provide values for the thermodynamic parameters that have been corrected for thermal fluctuations of the interacting molecules. In the temperature range from 13 degrees C to 45 degrees C (where all the binding reaction components are folded), formation of the complex is enthalpically driven with a negative heat capacity effect (DeltaC(p)). In this respect, the binding reaction of zf1-3 is similar to those of other proteins that bind in the major groove of DNA. It is dissimilar to the association reactions of proteins, however, that bind in the minor groove of DNA and that are driven by a dominating entropy factor. Comparison of the experimental values of DeltaH(ass) and DeltaC(p) with expected values of these parameters, calculated from the burial of polar and nonpolar molecular surfaces, indicates that the polar groups at the protein/DNA interface are not completely dehydrated upon formation of the complex. It also seems that the expected large positive entropy of dehydration upon forming the zfl-3/DNA complex ( approximately 1900 J * K(-1) * mol(-1)) cannot be balanced by the reduction in translational/rotational and configurational freedom of the protein to the level of the observed entropy of binding (38 J * K(-1) * mol(-1)). It is suggested that the additional negative entropy contribution comes from a damping of torsional motions in the DNA duplex.

Energetics of the specific binding interaction of the first three zinc fingers of the transcription factor TFIIIA with its cognate DNA sequence

The energetics of the specific interaction of a protein fragment (zf1-3) containing the three N-terminal zinc fingers of the Xenopus laevis transcription factor TFIIIA with its cognate DNA sequence, contained in a 15 bp DNA duplex were studied using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and fluorescence titration. The use of both ITC and DSC is necessary to provide values for the thermodynamic parameters that have been corrected for thermal fluctuations of the interacting molecules. In the temperature range from 13 degrees C to 45 degrees C (where all the binding reaction components are folded), formation of the complex is enthalpically driven with a negative heat capacity effect (DeltaC(p)). In this respect, the binding reaction of zf1-3 is similar to those of other proteins that bind in the major groove of DNA. It is dissimilar to the association reactions of proteins, however, that bind in the minor groove of DNA and that are driven by a dominating entropy factor. Comparison of the experimental values of DeltaH(ass) and DeltaC(p) with expected values of these parameters, calculated from the burial of polar and nonpolar molecular surfaces, indicates that the polar groups at the protein/DNA interface are not completely dehydrated upon formation of the complex. It also seems that the expected large positive entropy of dehydration upon forming the zfl-3/DNA complex ( approximately 1900 J * K(-1) * mol(-1)) cannot be balanced by the reduction in translational/rotational and configurational freedom of the protein to the level of the observed entropy of binding (38 J * K(-1) * mol(-1)). It is suggested that the additional negative entropy contribution comes from a damping of torsional motions in the DNA duplex.

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.

Increased heavy metal sensitivity of Escherichia coli producing the expression product of priA gene derived from the basidiomycete Lentinus edodes

We have previously isolated a developmentally regulated novel gene, priA, from the basidiomycete Lentinus edodes. The deduced PRIA protein contains the two set of motifs similar to a `zinc finger' typified by transcription factor TFIIIA and the motif of a `zinc cluster' observed in metallothioneins. It also contains a hydrophobic N-terminal sequence. Here Escherichia coli cells producing PRIA were found to show a remarkable sensitivity to zinc ion and other heavy metal ions such as nickel and cadmium. Deletion analysis of PRIA revealed that the zinc-binding motifs and the hydrophobic N-terminal sequence are responsible for conferring the heavy metal sensitivity on the host cells.

Increased heavy metal sensitivity of Escherichia coli producing the expression product of priA gene derived from the basidiomycete Lentinus edodes.

We have previously isolated a developmentally regulated novel gene, priA, from the basidiomycete Lentinus edodes. The deduced PRIA protein contains the two set of motifs similar to a 'zinc finger' typified by transcription factor TFIIIA and the motif of a 'zinc cluster' observed in metallothioneins. It also contains a hydrophobic N-terminal sequence. Here Escherichia coli cells producing PRIA were found to show a remarkable sensitivity to zinc ion and other heavy metal ions such as nickel and cadmium. Deletion analysis of PRIA revealed that the zinc-binding motifs and the hydrophobic N-terminal sequence are responsible for conferring the heavy metal sensitivity on the host cells.

DNA-protein cross-linking applications for chromatin studies in vitro and in vivo

Publisher Summary Covalent protein–nucleic acid cross-linking is a powerful tool for the analysis of protein–DNA and protein–RNA interactions in various biological systems. It allows a researcher to freeze protein–nucleic acid contacts within the context of a nucleoprotein complex and analyze cross-linked species by a variety of methods such as two-dimensional (2D) denaturing gel electrophoresis This chapter describes some of the methodical advances in the field of protein–nucleic acid covalent cross-linking applications. The innovations discussed include (1) a fast, relatively noninvasive method of cross-linking by radical-producing chemicals, such as bleomycin–iron and phenanthroline–copper complexes, (2) applications of chemical and photochemical cross-linking techniques to nucleosome-containing complexes following in vitro nucleosome reconstitution on specific DNA, and (3) a time-resolved cross-linking technique for studies of postreplicative chromatin assembly based on the immunoenrichment of newly replicated DNA. The chapter describes 5S rRNA gene nucleosome interactions with linker histones and transcription factor TFIIIA.

Enhanced transcription factor access to arrays of histone H3/H4 tetramer.DNA complexes in vitro: implications for replication and transcription.

Defined model systems consisting of physiologically spaced arrays of H3/H4 tetramer.5S rDNA complexes have been assembled in vitro from pure components. Analytical hydrodynamic and electrophoretic studies have revealed that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. The reptation in agarose gels of H3/H4 tetramer arrays is essentially indistinguishable from naked DNA, the gel-free mobility of H3/H4 tetramer arrays relative to naked DNA is reduced by only 6% compared with 20% for nucleosomal arrays, and H3/H4 tetramer arrays are incapable of folding under ionic conditions where nucleosomal arrays are extensively folded. We further show that the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 tetramer arrays.

Functionally Relevant Histone-DNA Interactions Extend Beyond the Classically Defined Nucleosome Core Region

We demonstrate that core histones can affect the accessibility of a DNA element positioned outside of the classically defined nucleosome core region. The distance between a well positioned nucleosome and the binding site for the 5 S-specific transcription factor TFIIIA was systematically varied and the relative binding affinity for TFIIIA determined. We found that core histone-DNA interactions attenuate the affinity of TFIIIA for its cognate DNA element by a factor of 50-100-fold even when the critical binding region lies well outside of the classically defined nucleosome core region. These results have implications for the validity of parallels drawn between the accessibility of general nucleases to DNA sequences in chromatin and the activity of actual sequence-specific DNA binding factors.

Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence.

The E1B 55-kDa and E4 34-kDa oncoproteins of adenovirus type 5 (abbreviated here as E1B-55kD and E4-34kD) promote the export of viral mRNA and inhibit the export of most cellular mRNA species. We show that the intracellular complex containing E1B-55kD and E4-34kD continuously shuttles between the nucleus and the cytoplasm, and may thus serve as a nucleocytoplasmic transporter for viral mRNA. We present evidence that within this complex, it is the E4-34kD protein that directs both nuclear import and nuclear export. E4-34kD contains a functional nuclear export signal similar to corresponding sequences found in the retroviral proteins rev and rex. This sequence element is required for nuclear export of the complex, and it can function autonomously when fused to a carrier protein and microinjected in HeLa cell nuclei. When E4-34kD is expressed alone, a portion of the protein that contains a predicted arginine-rich amphipathic alpha-helical structure mediates nuclear retention of the protein. This retention, however, can be abolished by the association with E1B-55kD or by a specific point mutation within the arginine-rich motif. The export of E4-34kD can be blocked by an HTLV-rex derived competitive inhibitor and overexpressed E4-34kD inhibits rev-mediated transport, suggesting that the export pathways accessed by the adenoviral and retroviral proteins share components. The interplay between two polypeptides as well as the involvement of a dominant nuclear retention domain are novel features that might contribute to the efficiency and regulation of the adenovirus export system.

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.

Molecular Cloning and Mapping of a Novel Human KRAB Domain-Containing C2H2-Type Zinc Finger to Chromosome 7q36.1

C2H2 zinc finger proteins constitute the largest family of transcription factors known with an estimated 300-500 genes encoding these proteins in the human genome. These proteins possess two conserved cysteines that are part of an antiparallel beta-sheet and two conserved histidines that are part of an alpha-helix, coordinated by a central zinc atom to form a globular domain. The protein family is typified by the RNA polymerase II transcription factor TFIIIA and the gap gene product Kruppel. 13 refs., 1 fig.

The only essential function of TFIIIA in yeast is the transcription of 5S rRNA genes.

We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A long transcript was synthesized both in vitro and in vivo from a hybrid gene in which the tRNA-like promoter sequence of the RPR1 gene was fused to the yeast 5S RNA gene. No internal initiation directed by the endogenous 5S rDNA promoter or any processing of the hybrid transcript was observed in vitro. Yeast cells devoid of transcription factor TFIIIA, which, therefore, could not synthesize any 5S rRNA from the endogenous chromosomal copies of 5S rDNA, could survive if they carried the hybrid RPR1-5S construct on a multicopy plasmid. In this case, the only source of 5S rRNA was the precursor RPR1-5S transcript that gave rise to two RNA species slightly larger than wild-type 5S rRNA. This establishes that the only essential function of TFIIIA is to promote the synthesis of 5S rRNA. However, cells devoid of TFIIIA and surviving with these two RNAs grew more slowly at 30 degrees C compared with wild-type cells and were thermosensitive at 37 degrees C.

The Interaction of TFIIIA with Specific RNA-DNA Heteroduplexes

We examine the association of transcription factor TFIIIA with RNA-DNA heteroduplexes containing sequences from the Xenopus borealis 5 S rRNA gene. Under conditions where TFIIIA selectively binds to 5 S rRNA or the internal control region of the 5 S rRNA gene, no specific association of TFIIIA with DNA-RNA heteroduplexes containing either strand of 5 S DNA could be detected. We discuss our results with respect to specific models of TFIIIA recognition of the internal control region and of DNA-RNA hybrids by zinc finger proteins.

Long-range motional restrictions in a multidomain zinc-finger protein from anisotropic tumbling.

Structural characterization of biomolecules in solution by nuclear magnetic resonance (NMR) spectroscopy is based primarily on the use of interproton distances derived from homonuclear cross-relaxation experiments. Information about short time-scale dynamics, on the other hand, is obtained from relaxation rates of heteronuclear spin pairs such as 15N-1H. By combining the two types of data and utilizing the dependence of heteronuclear NMR relaxation rates on anisotropic diffusional rotational tumbling, it is possible to obtain structural information about long-range motional correlations between protein domains. This approach was applied to characterize the relative orientations and mobilities of the first three zinc-finger domains of the Xenopus transcription factor TFIIIA in aqueous solution. The data indicate that the motions of the individual zinc-finger domains are highly correlated on time scales shorter than 10 nanoseconds and that the average conformation of the three-finger polypeptide is elongated.

Molecular cloning of a zinc finger protein belonging to the GLI-Krüppel family and its expression in the silk gland.

We have isolated and characterized a zinc finger-containing cDNA (ZSG-1) from the middle silk gland of Bombyx mori. Sequence analysis indicates that the protein encoded by the ZSG-1 transcript contains a domain of five fingers similar to the zinc finger repeats of the Xenopus transcription factor TFIIIA, the product of Drosophila segment polarity gene cubitus interruptus (ci) and the human zinc finger protein GLI, which has been found to be amplified in several human glioblastomas. The transcript expression pattern and protein distribution of ZSG-1 during embryogenesis and silk gland development were analyzed by northern blot and immunohistochemistry. ZSG-1 transcript was most strongly detected in the middle silk gland and weakly in the posterior silk gland at the fourth larval intermolt and molting stages. When the larvae entered the fifth intermolt, the expression of ZSG-1 rapidly decreased by 2 days after the fourth ecdysis and maintained a low level thereafter in the middle silk gland, while the transcript in the posterior silk gland gradually became undetectable. The transcript was first detected at around stage 18 of early embryogenesis; it then increased to the maximum level by stage 27 and maintained a high level until hatching. Interestingly, the ZSG-1 protein was most strongly distributed in the middle silk gland during embryonic silk gland development. The expression of ZSG-1 in the silk glands suggests that this gene may play important roles in the development of silk gland.