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During Herbert Tabor's tenure as Editor-in-Chief from 1971 to 2010, JBC has published many seminal papers in the fields of chromatin structure, epigenetics, and regulation of transcription in eukaryotes. As of this writing, more than 21,000 studies on gene transcription at the molecular level have been published in JBC since 1971. This brief review will attempt to highlight some of these ground-breaking discoveries and show how early studies published in JBC have influenced current research. Papers published in the Journal have reported the initial discovery of multiple forms of RNA polymerase in eukaryotes, identification and purification of essential components of the transcription machinery, and identification and mechanistic characterization of various transcriptional activators and repressors and include studies on chromatin structure and post-translational modifications of the histone proteins. The large body of literature published in the Journal has inspired current research on how chromatin organization and epigenetics impact regulation of gene expression., (© 2019 Gottesfeld.)

Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Basic helix-loop-helix (bHLH) transcription factors are characterized by a conserved four-helix bundle that recognizes a specific hexanucleotide DNA sequence in the major groove. Previous studies have shown that amino acids in the basic region make base-specific contacts, whereas the HLH region is responsible for dimerization. Structural data suggest that portions of the loop region may be proximal to the DNA; however, the role of the loop in DNA-binding affinity and specificity has not been investigated., Results: Protein-DNA recognition by the Drosophila bHLH transcription factor Deadpan was probed using combinatorial solid-phase peptide synthesis methods. A series of bHLH peptide libraries that modulate amino acid content and length in the loop region was screened with DNA and peptide affinity columns, and analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). A functional bHLH peptide with reduced loop length was found, and Lys80 was unambiguously identified as the sole loop residue critical for DNA binding. Unnatural amino acids were substituted at this position to assess contributions of the terminal amino group and the alkyl chain length to DNA-binding affinity and specificity., Conclusions: Using combinatorial solid-phase peptide synthesis methods and MALDI-MS, we were able to rapidly identify a key amino acid involved in DNA binding by a bHLH protein. Our approach provides a powerful alternative to current recombinant DNA methods to identify and probe the energetics of protein-DNA interactions.

Transcription factor IIIA of Xenopuslaevis serves a dual function during oogenesis and early development: this zinc finger protein binds to the internal promoter element of the 5 S ribosomal RNA genes and acts as a positive transcription factor; additionally, the protein functions in 5 S RNA storage. The central four zinc fingers (zf4-7) of the nine-finger protein have been shown to bind 5 S rRNA with comparable or higher affinity than the full-length protein. The role of finger seven in binding affinity has been examined by deletion analysis. A zf4-6 protein binds 5 S RNA with about a sevenfold reduction in binding affinity, compared to zf4-7. The effect of non-specific competitor DNA on binding affinities of the zinc finger peptides was examined and found to have a significant effect on the measured affinities of these peptides for full-length and truncated versions of 5 S RNA. The interaction of zf4-6 with full-length 5 S RNA was far more sensitive to non-specific competitor concentration than was the zf4-7:5 S RNA interaction, suggesting that finger seven contributes to both affinity and specificity in this protein:RNA interaction. In order to map zinc finger binding sites on the 5 S RNA molecule, we generated truncated versions of the RNA and tested these molecules for their binding affinities with zf4-7 and zf4-6. Previous studies showed that a 75 nucleotide long RNA, comprising loop A, helix II, helix V, region E and helix IV, bound zf4-7 with high affinity. Selection and amplification binding assays (selex) have now been used to generate smaller high-affinity binding RNAs. We find that a 55 nucleotide long RNA, comprising loop A, helix V, region E and helix IV, but lacking helix II, retains high affinity for zf4-6. These data are consistent with the proposal that fingers 4-6 bind this central core of 5 S RNA and that finger seven binds the helix II region., (Copyright 1999 Academic Press.)

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-kappaB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1. NF-kappaB.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-kappaB also inhibits NF-kappaB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

Transcription factor IIIB (TFIIIB) is composed of the TATA box binding protein (TBP) and class III gene-specific TBP-associated factors (TAFs). TFIIIB is brought to a site centered approximately 35 bp upstream from the transcription start site of tRNA genes via protein-protein interactions with the intragenic promoter-recognition factor TFIIIC. Since TBP interacts with TATA elements through the minor groove of DNA, we asked whether TFIIIB interacts with DNA in the minor groove. Polyamides containing pyrrole (Py) and imidazole (Im) amino acids are synthetic DNA ligands that bind to predetermined sequences in the minor groove of double helical DNA. These small molecules have been shown to interfere with protein-DNA interactions in the minor groove. A series of DNA constructs was generated in which the binding site for a Py-Im polyamide was placed at various distances upstream from a tRNA gene transcription start site. We find that a match polyamide will effectively inhibit tRNA gene transcription when its binding site is located within 33 bp of the transcription start site of the Xenopus TyrD tRNA gene. Moreover, in the presence of polyamide, RNA polymerase III is redirected to a new transcription initiation site located approximately one DNA helical turn downstream from the native start site. Our results suggest that a subunit of TFIIIB, possibly TBP, makes an essential minor groove DNA contact centered approximately 30 bp upstream from the tRNA gene., (Copyright 1999 Academic Press.)

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.

The gene-specific transcription factor IIIA (TFIIIA) binds to the internal promoter element of the 5 S rRNA gene through nine zinc fingers which make specific DNA contacts. Seven of the nine TFIIIA zinc fingers participate in major groove DNA contacts while two fingers, 4 and 6, have been proposed to bind in or across the minor groove. Pyrrole-imidazole polyamides are minor groove binding ligands that recognize predetermined DNA sequences with affinity and specificity comparable to natural DNA-binding proteins. We have examined the DNA binding activity of nine finger TFIIIA and shorter recombinant analogs in the presence of polyamides that bind six base-pair sequences (Kd = 0.03 to 1.7 nM) in the minor groove of the binding site for zinc finger 4. DNase I footprint titrations demonstrate that the polyamides and a recombinant protein containing the three amino-terminal zinc fingers of TFIIIA (zf1-3) co-occupy the TFIIIA binding site, in agreement with the known location of zf1-3 in the major groove. In contrast, the polyamides block the specific interaction of TFIIIA or zf1-4 with the 5 S RNA gene, supporting a model for minor groove occupancy by zinc finger 4. Minor groove binding polyamides targeted to specific DNA sequences may provide a novel chemical approach to probing multidomain protein-DNA interactions.

The high resolution solution structure of a protein containing the three amino-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound to its cognate DNA duplex was determined by nuclear magnetic resonance spectroscopy. The protein, which is designated zf1-3, binds with all three fingers in the DNA major groove, with a number of amino acids making base-specific contacts. The DNA structure is close to B-form. Although the mode of interaction of zf1-3 with DNA is similar to that of zif268 and other structurally characterized zinc finger complexes, the TFIIIA complex exhibits several novel features. Each zinc finger contacts four to five base-pairs and the repertoire of known base contact residues is extended to include a tryptophan at position +2 of the helix (finger 1) and arginine at position +10 (finger 3). Sequence-specific base contacts are made over virtually the entire length of the finger 3 helix. Lysine and histidine side-chains involved in base recognition are dynamically disordered in the solution structure; in the case of lysine, in particular, this could significantly decrease the entropic cost of DNA binding. The TGEKP(N) linker sequences, which are highly flexible in the unbound protein, adopt ordered conformations on DNA binding. The linkers appear to play an active structural role in stabilization of the protein-DNA complex. Substantial protein-protein contact surfaces are formed between adjacent fingers. As a consequence of these protein-protein interactions, the orientation of finger 1 in the major groove differs from that of the other fingers. Contributions to high affinity binding by zf1-3 come from both direct protein-DNA contacts and from indirect protein-protein interactions associated with structural organization of the linkers and formation of well-packed interfaces between adjacent zinc fingers in the DNA complex. The structures provide a molecular level explanation for the large body of footprinting and mutagenesis data available for the TFIIIA-DNA complex.

The three N-terminal zinc fingers of transcription factor IIIA bind in the DNA major groove. Substantial packing interfaces are formed between adjacent fingers, the linkers lose their intrinsic flexibility upon DNA binding, and several lysine side chains implicated in DNA recognition are dynamically disordered.

Differential transcription of the Xenopus gene families encoding the oocyte and somatic 5S ribosomal RNAs can be reproduced in vitro with cell-free extracts prepared from Xenopus oocytes and unfertilized eggs. The transcriptional activities of these genes as assayed in these in vitro systems are a consequence of large differences in the rates of assembly of active transcription complexes. The somatic 5S genes sequester limiting transcription factors much more rapidly than the corresponding oocyte 5S genes and, as a consequence, are far more active. However, once transcription complexes are formed, these complexes are stable on both of these genes. Previous studies have established that transcription factors IIIA and IIIC are sufficient to form a stable protein-DNA complex on the somatic 5S gene. The rate of formation of the stable TFIIIA+C complex for the oocyte gene is far slower than that for the somatic 5S gene. Insertion of the DNA binding site for TFIIIC2 (the B-block promoter element from tRNA genes) into the 3' flanking region of a synthetic oocyte 5S gene increases the transcription efficiency and rate of transcription complex assembly of this gene relative to the parent gene lacking the B-block element. Our results support a model in which competition for limiting transcription factors plays a pivotal role in establishing differential transcription of the two classes of 5S genes during early embryogenesis.

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 Xenopus 5 S ribosomal RNA gene promoter. Electron spectroscopic imaging analysis of TFIIIA:DNA complexes 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 degrees near the centre of the TFIIIA binding site. Helical phasing analysis demonstrates that the 60 degrees 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 degrees. 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.

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

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.

Derivatives of yeast tRNA and Xenopus tRNA and 5 S RNA genes have been constructed in which natural 5' flanking sequences have been replaced by the binding sites for either the yeast transcription activator protein GCN4 or the three amino-terminal zinc fingers of the Xenopus factor TFIIA (zf1-3). The binding sites for these proteins have been placed at various distances upstream from the start site for transcription initiation in the parent genes. Each of these plasmid DNAs is actively transcribed in both an unfractionated transcription extract prepared from unfertilized Xenopus eggs and in a reconstituted Xenopus transcription system. Binding of the test proteins to plasmid DNAs harboring the cognate binding sites severely represses transcription when these binding sites are located less than approximately 40 base-pairs upstream from the transcription start site. The DNA-binding proteins are without effect on the transcription of plasmids lacking binding sites or when the binding sites are located further upstream. Assembly of DNA templates into a complete transcription complex prior to addition of the DNA-binding proteins prevents repression. Proteins present in a fraction containing TFIIIB are necessary for this reversal of repression. These data suggest that vertebrate TFIIIB binds upstream from class III genes and this binding can be prevented by occlusion of the TFIIIB binding site by the test proteins GCN4 and zf1-3.

Zinc fingers 4 to 7 of Xenopus transcription factor IIIA (TFIIIA) represent the minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for their effects on the binding affinity of full-length TFIIIA and a polypeptide consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease footprinting was used to compare the binding sites of TFIIIA and zf4-7 on 5 S RNA. The consistency between the data obtained from these two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA bind to a central core region on the 5 S RNA molecule consisting of loop B/helix II/loop A/helix V/region E. This information was used to design a truncated 75-nucleotide-long RNA molecule that retains high affinity for zf4-7. Therefore, we conclude that the specific interaction of TFIIIA with 5 S RNA can be represented by a complex formed between a four zinc finger polypeptide and a truncated 5 S RNA molecule.

We have expressed and purified a series of recombinant zinc finger polypeptides derived from the cDNA for the Xenopus 5 S gene-specific transcription factor TFIIIA. Dissociation constants for the interaction of each of the truncated polypeptides with the 5 S gene promoter have been measured using gel mobility shift assays. DNase I footprinting and proteolysis experiments provide additional insights into the interactions of individual fingers within complexes of the truncated proteins. These results are discussed in terms of recently proposed models for the TFIIIA-DNA interaction. The effects of mutations in two of the strongly binding proteins, zf1-3 and zf1-7, on DNA binding affinity have been investigated. Mutations have been made both in putative DNA-contact residues and in the linker regions between zinc fingers. The observed decreases in binding affinity cannot be explained simply in terms of loss of protein-DNA contacts. Our results support a model in which DNA binding is accomplished through sets of interacting zinc fingers that make different energetic contributions to the overall binding of the protein and different contacts with the DNA.

Interphase cytosol extracts prepared from Xenopus laevis eggs are active in RNA polymerase III (Pol III) transcription. Addition of recombinant B1 cyclin to these extracts activates mitotic protein kinases that repress transcription. Affinity-purified p34cdc2-cyclin B kinase (mitosis-promoting factor) is sufficient to effect this repression in a simplified Pol III transcription system. This mitotic repression involves the direct phosphorylation of a component of the Pol III transcription initiation factor TFIIIB, which consists of the TATA box-binding protein (TBP) and associated Pol III-specific factors. The transcriptional activity of the TFIIIB-TBP fraction can be modulated in vitro by phosphorylation with mitotic kinases and by dephosphorylation with immobilized alkaline phosphatase.

Transcription factor IIIA (TFIIIA) from Xenopus oocytes binds both the internal control region of the 5S ribosomal RNA genes and the 5S RNA transcript itself. The nucleic acid binding domain of TFIIIA contains nine tandemly repeated zinc finger motifs. A series of precisely truncated forms of this protein have been constructed and assayed for 5S RNA and DNA binding. Different sets of zinc fingers were found to be responsible for high affinity interactions with RNA and with DNA. These results explain how a single protein can exhibit equal affinities for these two very different nucleic acids.

A series of polypeptides containing increasing numbers of zinc fingers of Xenopus transcription factor IIIA has been generated and binding to the 5S RNA gene internal control region has been studied in order to elucidate the mode of interaction of the individual fingers with DNA. By using a combination of DNase I footprinting, methylation interference, and differential binding to mixtures of DNA fragments differing in length by single base pairs, the binding sites for individual fingers have been defined. These results have led to a model for the interaction of transcription factor IIIA with the internal control region in which fingers 1-3 bind in the major groove of the promoter C block, fingers 7-9 bind in the major groove of the A block, and finger 5 binds in the major groove of the intermediate element. Fingers 4 and 6 each bind across the minor groove, spanning these promoter elements.

The general transcription factor TFIIIC is necessary for transcription initiation by RNA polymerase III. TFIIIC binds predominantly to the B-Block promoter element, which is present in tRNA genes, several viral RNA genes and repetitive DNA elements, and to the TFIIIA.DNA complex on 5 S RNA genes. Here we report a characterization of Xenopus laevis TFIIIC and its interaction with the TFIIIA.5 S RNA gene complex. A polypeptide with apparent molecular mass of 85 kDa was specifically cross-linked to a B-Block oligonucleotide by UV light. This polypeptide was present in the partially purified TFIIIC fraction and in a complex with a B-Block double-stranded oligonucleotide isolated by nondenaturing gel electrophoresis. TFIIIC.TFIIIA.DNA gel mobility shift complexes were obtained using B-Block DNA affinity-purified TFIIIC and buffer conditions employing low Mg2+ (1 mM) and high dithiothreitol (7 mM) concentrations. Three TFIIIC.TFIIIA.5 S RNA gene complexes were observed by gel mobility shift analysis. One of these complexes was resistant to dissociation by the addition of competing DNA, but the formation of all three complexes was prevented by the inclusion of excess specific competitor DNA in the initial binding reactions. The apparent affinity of TFIIIC for the TFIIIA.5 S DNA complex was 5-fold higher for the somatic-type 5 S RNA gene than for the oocyte-type 5 S RNA gene. Mutations near the 5' boundary of the TFIIIA binding site alter the DNase I footprint of the TFIIIA.DNA complex and reduce the affinity of TFIIIA-mutant 5 S gene complexes for TFIIIC. Differences in TFIIIC affinity for the two classes of 5 S RNA genes may play a role in the developmental regulation of these gene families.

A DNA plasmid encoding the first 101 amino acid residues of the Xenopus 5 S RNA gene-specific transcription factor IIIA (TFIIIA) was derived by polymerase chain reaction amplification of this region from the cDNA for TFIIIA. This polypeptide includes the first three zinc fingers of the TFIIIA DNA binding domain. The polypeptide was expressed in Escherichia coli and purified to greater than 95% homogeneity. The three finger polypeptide binds the internal control region of the 5 S RNA gene with sequence specificity and high affinity. Binding is metal-dependent and treatment of the polypeptide with EDTA abolishes binding. Polypeptide-DNA complexes exhibit a dissociation constant of 5.6(+/- 0.9) nM, while that for full-length Xenopus TFIIIA is 2.2(+/- 0.4) nM, measured under the same conditions. This suggests that the majority of the free energy of TFIIIA binding resides in these amino-terminal zinc fingers. The polypeptide protects 21 base-pairs of the internal control region from attack by DNase I, with protection from nucleotides +75 to +95 of the 120 base-pair gene. This region includes the C-block promoter element and several guanine residues that are essential for TFIIIA binding. Methylation interference experiments suggest that the mode of binding of the polypeptide and TFIIIA are similar. The minimal DNA sequences required for polypeptide binding were determined using a series of synthetic double-stranded deoxyribo-oligonucleotides. A 13 base-pair oligonucleotide spanning nucleotides +80 to +92 of the 5 S RNA gene retained specific and high-affinity binding, although the latter was reduced sixfold relative to longer DNA fragments. Polypeptides containing fingers 1 and 2, or fingers 2, 3 and 4 of TFIIIA do not exhibit sequence-specific DNA binding. Overall, these studies provide strong support for a model in which the first three zinc fingers of TFIIIA bind with high affinity between nucleotides +80 and +92 of the internal control region of the 5 S RNA gene.

We have used a circular permutation gel shift assay to show that the 5S gene transcription factor, TFIIIA, induces a bend at the internal promoter of the Xenopus oocyte-type 5S gene. The degree of bending is comparable to what we have previously observed for TFIIIA induced bending of the Xenopus somatic-type gene [Schroth, G.P. et al. (1989) Nature 340, 487-488]. In addition, we show that TFIIIA induced DNA bending is dramatically affected by the ionic conditions used during gel electrophoresis. By modifying the conditions of the electrophoresis, we can detect two distinct conformations for the TFIIIA/DNA complex. In very low ionic strength buffers, the degree of DNA bending in the complex is estimated to be about 25 to 30 degrees, whereas in higher ionic strength buffers it is about 60 to 65 degrees. These data explain the apparent discrepancy between our results and the results of another study in which it was claimed that TFIIIA did not 'substantially' bend DNA [Zweib, C. and Brown, R.S. (1990) Nucleic Acid Res. 18, 583-587]. These results also demonstrate that the TFIIIA/DNA complex has a large degree of conformational flexibility. Both DNA bending and conformational flexibility are structural features which may provide a key insight into the function of TFIIIA as a positive transcription factor.

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.

Accurate transcription of eukaryotic genes in vitro is preceeded by the assembly of the template DNA into stable DNA-protein complexes. Such complexes have been reported for genes transcribed by each of the three eukaryotic RNA polymerases. DNAase I protection or footprint assays have yielded information as to the sites of protein factor binding. These sites correlate with many of the sequences which have been implicated as promoter elements through analysis of deletion mutants. Stable transcription complexes are also formed in microinjected Xenopus oocytes and such complexes can be shown to exist in vivo in nuclei and chromatin. The propagation of active transcription complexes may prove to be a crucial element in cellular differentiation.

We have measured the transcription of Xenopus laevis oocyte and somatic-type 5 S RNA genes in S-150 extracts prepared from ovaries of mature X. laevis females (Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Cell 37, 33-41). We find that somatic-type 5 S genes are transcribed at least 40-fold more efficiently than oocyte-type 5 S genes. Since adenovirus VA, Xenopus OAX, and Xenopus tRNAMet genes are all transcribed at levels similar to that of the somatic-type 5 S gene, this difference reflects a low level of oocyte-type 5 S gene transcription. Somatic-type 5 S transcription is a linear function of somatic-type 5 S DNA concentration and this, together with the efficient transcription of other class III genes, suggests that RNA polymerase III and the general class III transcription factors are not limiting under the conditions used here. Moreover, the 5 S gene-specific transcription factor TFIIIA does not limit transcription under these conditions as preincubation with purified TFIIIA does not alter transcription of either gene, and both genes exhibit characteristic TFIIIA footprints under transcription conditions in the S-150. Somatic-type 5 S DNA incubated in the S-150 and then injected into oocyte nuclei is actively transcribed whereas oocyte-type 5 S DNA treated in the same manner is inactive. We conclude that factors in the S-150 distinguish between somatic and oocyte-type 5 S genes, assembling active complexes preferentially on the former and inactive complexes preferentially on the latter. This process is not explained by binding properties of transcription factors for these genes as currently understood and represents a previously unrecognized mechanism of transcriptional regulation of the Xenopus 5 S genes.

In vitro transcription studies of cloned messenger RNA-coding genes have yielded considerable information regarding the sequence elements and protein factors involved in transcription initiation and RNA processing. Fractionation of whole-cell, S-100 protein and nuclear extracts reveals the existence of both general class II and gene-specific transcription initiation factors. Because the soluble in vitro transcription systems prepared from cells in culture are largely nonspecific for the origin of the template DNA, they are highly suited to searching for tissue-specific and gene-specific transcription regulatory factors. In the experiments reported here, we have added a nuclear extract prepared from human erythroleukaemia-like cells (K562, which can be induced to synthesize epsilon- and gamma-globin mRNA and protein) to several deproteinized DNA templates, and monitored transcription levels in a HeLa cell-free transcription system. The K562 nuclear extract enhanced transcription of beta-, epsilon- and gamma-globin genes by as much as 30-fold compared with control non-globin templates. These results suggest the presence of a globin gene regulatory factor in erythroleukaemia cell nuclei.

Rhodes and Klug have recently proposed that the internal control region of the Xenopus 5S RNA gene adopts an A-type DNA structure in solution. This suggestion was based on a Fourier analysis of both the spacing of DNase I cutting sites and on the distribution of G residues in the DNA sequence. Both revealed a approximately 5.6-5.7-base periodicity which the authors interpreted as a structural repeat every half helical turn of A-type DNA. This contention was strengthened by the finding that a 9-base-pair (bp) double-stranded deoxyoligonucleotide corresponding to residues +81 to +89 of the 5S gene exhibits an A' RNA-like crystal structure. This region of DNA is of special interest as it forms the binding site for the 5S gene-specific transcription factor IIIA (TFIIIA). TFIIIA is a Zn2+-binding protein which interacts with both the internal control region of the gene and the 5S transcript. As base-paired regions of RNA are of the A type, it was reasonable to postulate that 5S DNA might also adopt this conformation. We report here that the circular dichroism (CD) spectrum of a synthetic 54-bp deoxyoligonucleotide corresponding to the TFIIIA binding site is similar to the CD spectrum of B-form DNA in solution. Further, DNA-TFIIIA complexes show an unaltered DNA CD component indicating no gross alteration in DNA structure on protein binding.

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

Relaxation of Xenopus 5S plasmid DNA (pX1o8) in the presence of transcription factor (TF) IIIA reduces the linking number of the DNA. Parallel experiments with plasmid pMB9 or cloned hepatitis B viral DNA indicate a degree of non-specific unwinding by TF; however, 60% of the effect observed for pX1o8 is due to specific interaction of TF IIIA with the 5S rRNA gene internal promoter sequence. The extent of unwinding (0.2-0.4 helical turn per TF IIIA binding site) is not consistent with the complete denaturation of the 50-base-pair TF binding site; however, it is consistent with a change in helix rotation, denaturation of 2-4 nucleotides per binding site, or DNA wrapping about a protein core. We show that proteins other than TF IIIA (bovine serum albumin and RNase) have no effect on the linking number of DNA when present during relaxation and that the unwinding activity associated with TF is heat labile. These results suggest that TF IIIA may facilitate transcription by altering the helical configuration of 5S DNA.

Transcription factor IIIA (TFIIIA), the canonical zinc-finger protein, is a protein of relative molecular mass 39,000 (39K) that is required for transcription of 5S-ribosomal subunit genes in Xenopus. It binds in a sequence-specific manner to the internal control region of the 5S gene (see Fig. 1) and facilitates transcription of the gene by RNA polymerase III. It also binds to the 5S gene product to form a 7S ribonucleoprotein particle. In oocytes the 7S particle acts as a storage form of the RNA to be utilized later in development. TFIIIA binds to DNA through its 30 K N-terminal domain, which contains nine zinc-fingers. TFIIIA was the first protein described to have this type of DNA binding motif, but numerous other proteins have now been shown to have zinc-finger domains. A structure for a single zinc-finger from the yeast protein ADR1, was recently proposed based on two-dimensional NMR data (ref. 8), and a similar structure was proposed based on comparison with crystal structures of other metalloproteins. Although models for the interaction of TFIIIA with the 5S-ribosomal gene DNA have been proposed, based on nuclease digestion and methylation interference data, little precise structural information is available for TFIIIA and the physical basis for the interaction of zinc-fingers with DNA is not understood. Using both circular permutation and circularization assays we provide convincing biochemical evidence that TFIIIA bends the DNA at the internal promoter of the 5S gene.

The 5S gene-specific transcription factor TFIIIA forms an exceptionally stable complex with the internal promoter of the 5S RNA gene. Approximately 1 to 5% of TFIIIA-DNA or deoxyoligonucleotide complexes are stable to harsh denaturation conditions and can be resolved by electrophoresis in the presence of SDS. These complexes are resistant to acidic conditions (0.1 N HCl, 55 degrees C, 2h) suggesting that the interaction may be through a covalent bond. Complex formation does not result in DNA strand scission and studies of the chemical sensitivity of the complex suggest that the TFIIIA-DNA linkage may be through a phosphoramidate bond. Covalent complexes are formed with both the noncoding (RNA-like) and coding strands of the internal control region. The predominant sites of TFIIIA-DNA adducts have been mapped to the 3' end of the 5S gene internal control region, the region previously shown to exhibit essential guanine and phosphate contacts with TFIIIA.