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Volume 14 Number 11 1986 14 Number 11 1986 Volume Nucleic Acids Research Nucleic Acids Research TFIIIA and homologous genes. The 'finger' proteins Alain Vincent Institut Jacques Monod, CNRS and Universite Paris VII, 2, place Jussieu, 75251 Paris Cedex 05, France Received 3 April 1986; Accepted 2 May 1986 ABSTRACT Differential regulation of gene expression , in a precise temporal and spatial pattern during development, is thought to be partly mediated by site specific DNA binding proteins which promote a selective activation of qene transcription (1). From studies on XenopusTFl IA, a factor selectively required for transcription of 5 S ribosomal RNA genes, Miller etal. (2) proposed a novel structural model of interaction between DNA and DNA binding protein. The striking homology of TFI I IA with several recently sequenced Drosophi/aand yeast gene products suggests that multiple regulatory proteins may have evolved from a small ancestra DNA binding protein domain and that the characteristic features of TFIIIA and TFIIIA-5S DNA interactions may be of general significance. TFI I IA and the develoDmental control of 5 5 RNA gene exDression in XenoDus 5 5 ribosomal RNA gene transcription In Xenopus provides a model system for studying differential gene regulation during development. Initiation of transcription of 5 S genes by polymerase III requires the formation of a "stable transcription complex" including DNA in the internal control region (ICR) and at least three transcription factors, TFIIIA, B and C (3), the specific Interaction between TFIIIA and the internal control region being the primary event in the activation of the genes (4). The establishment of stable active complexes within the otherwise transcriptionally silent chromatin has been postulated to provide a means for maintaining the selective expression of a specific set of activated genes; the repressed genes not complexed with transcription factors are prevented from recruiting these factors by a chromatin structure dependent on histoneHI (5). Both oocyte type and somatic type 5 S RNA genes (20 000 and 400 genes per haploid genome, respectively) are transcribed during oogenesis. In somatic cells, however, more than 95% of the 5 S RNAs are transcribed from the somatic type genes, which corresponds to a greater than 1000 fold preferential transcription of somatic over oocyte type genes on a per gene basis. Much of this preference for somatic 5 S gene transcription is attributed to the concentration of the positive © I R L Press Limited, Oxford, England. 4385 Nucleic Acids Research transcription factor, TFIIIA, and to differences in binding constant of TFIIIA to the 50 bp long ICR of the two types of genes (the two ICR differ by 3 bp ) (6). TFIIIA is a single protein of 38500 dalton which interacts also with 5 S RNA to form 7 S ribonucleoprotein particles that is a storage form of 5 S RNAs during oogenesis. Early in oogenesis, TFIIIA is present in excess, ensuring thereby the activation of both types of 5 S RNA genes. As the cellular concentration of TFIIIA decreases during late oogenesis and embryogenesis it becomes a limiting factor. This results in the preferential formation of stable active complexes with somatic 5 5 genes. The imposition of complete oocyte 5 S gene repression could occur gradually over a few cell divisions (7). Binding of TFI II A to the 5S RNA gene: "the finger Drotein model". The intact TFI I IA factor binds to the ICR as a monomer and protects about 50 nucleotides in the center of the 5 5 RNA gene from digestion by DNAse 1. Three functional domains are separable by proteolytic cleavage. At the C terminus of the protein is a domain required for efficient RNA transcription but not for binding to DNA. A second domain binds to the 5' end of the ICR, and, together with the third which binds only to the 3' side of the control region but Is Inactive In promoting transcription, partly supports transcription (8). Computer analysis of the predicted TFIIIA aminoacid sequence (9) revealed a continuous run of nine similar units, each of about 30 residues (residues 13 to 276), covering the ICR binding domain (2,10). The sequence information, the high zinc content of the 75 ribonucleo- protein particle, and more detailed studies on proteolytic digestion led Miller et 8f (2) to propose that TFIIIA structure includes nine loop like domains, the "fingers", formed by interaction of two invariant pairs of cysteines and histidines with an atom of zinc. The key feature of the suggested structure Is that the "fingers" (schematically drawn on figure 1B), which are the proposed DNA binding regions, are independent units linked by flexible joints; the Phe and Leu residues may form a hydrophobic core within each finger, and the fingers may bind to nucleic acids by interaction of positively charged DNA-binding amino acids with the phosphate backbone of DNA (1 1). Such a structure, consistent with the asymetry of the TFIIIA DNA binding site, could explain : 1) how a relatively small protein could bind to a long stretch of double helical DNA, because each "finger" interacts with roughly half a DNA period, and 2) how a "stable" transcription complex is able to wlthstand the continuous cycling of RNA polymerase through it because some fingers remain in contact with the DNA while others release this contact as polymerase passes by. A three dimensional model of a triple complex formed between TFIIIA, the histone octamer and the 5S RNA gene is further proposed by Rhodes (12) to explain how RNA polymerase III 4386 Nucleic Acids Research a 1 Xen.TFINA Dros.Kr Dros. sry 3 6 10 .0. .© . 16 23 19 29 ®.. . ® -<13 0* e ........)® C)..e.©..C 8)® .(®* c). Yeast ADRI D.D 0 © . .* . . . . - --*c- *(o *60 09 .(. . - ® *C)...(®0. .(D - . .. 29 a.a. - - - - . so* .® . * 30a.a. 28 a.a. . ®( 28 a.a. b Zn Xen. Dros. Figure 1. Alignment of repeats present in the TFIIIA (ref.2), Ar (ref.14), beta and delto (ref.13), and ADRI (ref.30) polypeptides is shown. The second sr,y csteine of the cysteine pair is arbitrarily placed at position 6 of the repeat. Onl the best conserved residues are indicated. The potential DNA-binding loop (finger) is formed by residues 7 to 18 indicated bu larger black dots (see ref.2). Folding scheme for the A17nopilu,s and Drzisopbi/ repeated domains: the finger structure, drawn according to the model proposed bu Miller et al. (2). Each domain is centered on a tetrahedral arrangement of metal (Zn) ligands. The marked residues are the conserved amino acids which include the Cys and His metal ligands and the three hydrophobic residues that may form a structural core. could interact simultaneously with transcription factors bound at the internal control region of the gene and the start point of transcription and how histone HI could repress transcription of 5S RNA genes by preventing TFIIIA binding. Prosobih,g fingeproteins. Sequence analysis of the Drosopbe/5 genes Serendipity (s.rg) beta and delta (13), and Kruppel (At-) (14) revealed that the finger motif has been conserved during evolution. Figure IA shows the characteristic features of the 30 residues TFIIIA repeated domain preserved in the DrosopbiJ'A repeat- the Cys 3,Cys 6, Phe 10, Leu 16, His 19 and His 23 - , i.e., the precise spacing between Cys 6 and His 19, the putative DNA binding domain. The Cys-X2-Cys motif and additional Cys and His residues are found at regularly spaced positions in the DNA binding region of various nucleic acids binding proteins whith no close homology with TFIIIA. . Examples are proteins encoded by the gag-pol region of retroviruses, the [rcsqsop4l/y copia element and the cauliflower mosaic Yirus (15,16), as well as the recently sequenced human and avian cestrogen receptors and the human glucocorticoid receptor (17,20). In the latter examples, three Cys-X2-Cys units and additional Cys and Lys residues are 4387 Nucleic Acids Research found at equivalent positions in a 66 amino acids domain -the putative DNA binding site- highly homologous between the three proteins. This suggests that the DNA binding domain of these proteins may include one or more independent folding units, but markedly different from the TFIIIA finger (20). The metallothionein family of proteins represents another group of proteins which contain repeated Cys residues (21), but is not a good analogy forTFIIIA (2). The extensive homology of Drosopbil5 s,rg beta, sr,y delta and At with TFI I A raises the strong possibility that -.r,y and A', code for DNA (or RNA) binding proteins whith some properties similar to those of TFIIIA. This is of considerable interest since the Ar gene falls within the class of Dr.csap4lJ/M segmentation genes required for the establishment of adjacent groups of segments (22) and presumably acts on the expression of other (sets of) specific genes (14). No obvious morphological defect has yet been associated with mutations at the sry complex locus; embryos carrying a delta mutation over a deficiency die before hatching (J. Lengyel, personal communication). It is then remarkable that a yeast regularory protein, ADRI, which is required for the transcriptional activation (derepression) of the alcohol dehydrogenase (ADH2) gene also shows amino acid sequence homology with the repeated domain of TFI I IA ((30), see fig. 1A). Two fingers are found in the amino terminal third of ADRI, a domain which is as active as the entire protein in ADH2 derepression. 4, 6 and 7 "fingers" are found in the Kr7 sri' beta and sry delta polypeptides, respectively (figure 2). One complete finger has been found in yet another Drosqopfl/l blastoderm specific gene (J. Lenggel, personal communication ). The finger region is the only region of homology between the Drosopbi/8 finger proteins, suggesting that partial and independent gene duplication and/or conversion events have been at the origin of finger protein coding genes. Within the A;:7 ;r/& beta and ,y delta polypeptides, each "finger tip" displays a unique sequence of "DNA binding amino acids". This suggests that the contacts between DNA and these proteins do probably not involve a stretch of repeated short sequences (see, for example, the 5S-TFIIA contact region). Although beta and delta most likely arose from a duplication event after the multiplication of the internal repeat the degree of conservation between fingers at homologous positions in the beta and delta proteins is variable. This might reflect unequal evolutionary constraints on the different fingers perhaps because DNA recognition and DNA binding strength are not carried by the same and all fingers. The invariance of the amino acid stretch between His 23 and Cys 3 (the inter-finger region, Fig. 1) in Kr suggests that this portion of the repeat is important for the folding of that protein. The portions of the TFIIIA, Kr or srb, 4388 Nucleic Acids Research TFIIIA 000 0 0 1H_2 Kr NH 2 2COOH 2 33~2 ~~~~222 1 NH 00 0 sry beta 344 0 0 529 CH 0 166351 1 sry delta N1 193 4 3 ADR i 1 98 (1' " 5155 NH8 1323 COOH Figure 2. Scheme for a linear arrangement of fingers in the TFIIIA (344 a.a.), A (529 a.a.), srg beta (351 a.a.), s,y delta (430 a.a.) and ADR1 (1323 a.a.) polypeptides. Orientation of each polypeptide is amino (left) to carboxy terminus polypeptides which are adjacent to the finger region and are characteristic of each polypeptide might in turn be involved in specific protein-protein interactions. DISCUSSION The finding that several Drasap/Ai/ and one yeast protein contain a repeated "finger" motif supports the prediction (2) that various regulatory proteins may have evolved from a small DNA binding ancestral domain. Furthermore, it predicts that variations in the DNA (or RNA) binding strength of each finger -by variations in the sequence at each finger tip- and in the number of fingers could establish a highly specific recognition system for nucleic acid segments. Such a system of DNA recognition is clearly different from the classical bacterial system - a DNA binding-domain made of two alpha helices (CZ' and "3) connected by a beta turn with alpha helix '3" lying into the major groove of DNA (23), or the presumably analogous system in eucaryotes, carried by regulatory protei-ns such as the yeast- mat a 1 and alpha 2 and the Drosop,t'// homeo-box containing gene products (24,25). Miller at &I (25) propose that eucaryotic DNA-binding proteins which recognise the same DNA sequence , such as mat a 1 4389 Nucleic Acids Research and alpha 2 or, possibly, several homeo-domain containing OrosopbfAie proteins, could perform different functions because of the different protein-protein interactions they can make. The finger proteins may constitute a separate class of regulatory proteins. Their characteristics may include the variable length and asymetry of their DNA binding sites, (their binding sites might often lie within the transcribed DNA (enhancers?), multiple fingers circumventing the passing polymerase problem) the I to 1 stoechiometry of binding, and the possibility to induce DNA gyration and form dynamic chromatin, a reaction that appears to be the key step -formation of dynamic chromatin- in the activation of the 5S RNA gene (26). DraSOpA//i "finger' proteins and development; chromosomal regulation of gene expression? The Kruppel gene function is required for the formation of thoracic and abdominal segments in the DrmopsV1/Ae embryo. Kr gene expression is strictly zygotic, and restricted to the blastoderm and early gastula stages in the portions of the embryo corresponding to the anlage of the thoracic and anterior abdominal segments missing in a strong Ar mutant (27). SIry gene expression is also developmentally regulated. Sry beta and delta mRNA accumulate during oogenesis, are abundant in polysomes of early embryos but (similarly to TFIIIA RNA in A%inqp/s) are present at a very reduced level during the rest of the fly life cycle (a). Pattern formation and cell differentiation are thought to result from the coordinate expression of specific combinations of regulatory genes. The combinatorial aspect of control might be reflected at the level of chromatin structure by the fact that a given gene, depending on the cell looked at, displays a particular array of DNase I hypersensitive sites which are thought to correlate with the presence of trans-acting control elements (29). From the data summarised above, and with reference to the concept of chromosomal regulation of gene expression (7,29), it is tempting to propose that 'finger" proteins include RNA pol ii associated positive transcription factors whose mode of action is analogous to that of TFIIIA. These factors would act primarily by antagonising the binding of histone HI to the chromatin and promoting the selective formation of "activated transcription complexes" at specific gene sites thus 'determining these genes. Autocatalytic activation of a gene coding for a positive transcription factor might then depend on the affinity of the protein for its own gene control region. Finally, exists the interesting possibility that finger proteins other than TF IIA are capable of binding to specific RNA, and mediate an autoregulation of gene expression (14). 4390 Nucleic Acids Research The multi-finger structure of the proteins considered here may be typical of many others present in eucearyotic cells that recognise specific DNA sequences and help to determine the temporal and spatial pattern of gene expression during development. Isolation of several such proteins might represent an important step towards the understanding of how DNA binding proteins act to regulate gene expression. 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