May 15, 1995 - performed as described in Webster et al. (1988). Each transfection experiment was done in triplicate, with at least two different plasmid.
The EMBO Journal vol.14 no. 15 pp.3777-3787, 1995
Staf, a novel zinc finger protein that activates the RNA polymerase III promoter of the selenocysteine tRNA gene Catherine Schuster, Evelyne Myslinski, Alain Krol and Philippe Carbon1 UPR 9002 du CNRS 'Structure des Macromolecules Biologiques et Mecanismes de Reconnaissance', IBMC, 15 rue Rene Descartes, 67084 Strasbourg Cedex, France
'Corresponding author The selenocysteine tRNA gene (tRNAse) is atypical. Though transcribed by RNA polymerase III like all other tRNA genes, its basal promoter elements are distinct and reside essentially upstream of the coding region. In addition, transcription from the basal promoter is activated by a 15 bp activator element. In this report we describe the cloning and functional characterization of Staf (selenocysteine tRNA gene transcription activating factor), a novel Xenopus laevis transcription factor which binds to the tRNASeC activator element and mediates its activation properties. The 600 amino acid Staf protein contains seven zinc fingers and a separate acidic activation domain. Seven highly conserved regions were detected between Staf and human ZNF76, a protein of unknown function, thereby aiding in predicting the locations of the functional domains of Staf. With the use of a novel expression assay in X.laevis oocytes we succeeded in demonstrating that Staf can activate the RNA polymerase III promoter of the tRNAsec gene. This constitutes the first demonstration of the capacity of a cloned factor to activate RNA polymerase III transcription in vivo. Keywords: RNA polymerase III/selenocysteine/transcriptional activator/tRNAsec gene/zinc finger
Introduction tRNA selenocysteine (tRNASec) is the carrier molecule upon which selenocysteine is synthesized and serves as a donor of selenocysteine to nascent selenoproteins in response to specific UGA selenocysteine codons (for reviews see Hatfield et al., 1990; Baron and Bock, 1995). The basal promoter of the RNA polymerase III (Pol III) tRNASec gene is tripartite (Carbon and Krol, 1991). It comprises two upstream promoter elements, a proximal sequence element (PSE), a TATA motif and an intragenic element, the B box, as in other tRNA genes. The tRNASec gene is therefore a representative of a class of Pol III genes with mixed promoters (for reviews see Gabrielsen and Sentenac, 1991; Willis, 1993). Curiously, this tRNA gene does not possess the internal A box which, in Pol III genes with internal promoters, is thought to be an obligatory element to convey the effect of the B box, via TFIIIC (for a review see Geiduschek and Kassavetis, 1992). The PSE and the TATA motifs of the tRNASec gene are functionally equivalent to K Oxford University Press
those of vertebrate U6 snRNA genes. The U6 snRNA gene is the prototype of a subclass of several Pol III genes that contain external upstream promoters but no intragenic control regions (Carbon et al., 1987; Krol et al., 1987; Mattaj et al., 1988; Lobo and Hernandez, 1989; for reviews see Gabrielsen and Sentenac, 1991; Kunkel, 1991; Geiduschek and Kassavetis, 1992; Hernandez, 1992; Willis, 1993). It looks as though the transcription complex which assembles on the tRNASec basal promoter comprises transcription factors common to vertebrates U6 genes. Indeed, we have recently shown that TBP (TATA binding protein) and PBP (PSE binding protein) are required for transcription in vitro from the tRNASec promoter (Myslinski et al., 1993a; Meissner et al., 1994), much as the U6 promoter (Simmen et al., 1991; Waldschmidt et al., 1991; for a review see Hernandez, 1992, and references therein). In contrast to tRNA genes with internal promoters, TBP is recruited to the tRNASec promoter via direct DNA contacts at the TATA box (Myslinski et al., 1993a). Pol III genes with external or mixed promoter elements contain, in addition to the cis elements described above, a distal element which possesses the property of augmenting the transcription level (Carbon et al., 1987; Kunkel and Pederson, 1988; Kleinertetal., 1990, Myslinski etal., 1992; Danzeiser et al., 1993; for a review see Hernandez, 1992). In the Xenopus U6 gene this distal sequence element (DSE) is composed of two motifs consisting of an octamer (Carbon et al., 1987, Krol et al., 1987) and an SpI motif (Lescure et al., 1992a), which binds Oct-I and SpI proteins respectively. In the Xenopus laevis tRNASec gene the distal activator element, termed AE, contains one single 15 bp CCAGCATGCCTCGCG motif residing between -209 and -195 (Myslinski et al., 1992). It was shown earlier to bind a nuclear protein in vitro and function in a self-sufficient fashion, since it does not require the assistance of other motifs, in particular the octamer (Myslinski et al., 1992, 1993b). Here we describe the cDNA cloning and functional characterization of Staf, a novel X. laevis transcription factor which binds specifically to the AE and mediates its activation properties. For this purpose we developed an expression assay in which staggered micro-injections of Staf mRNA and reporter genes into the cytoplasm and nucleus respectively enabled Staf to be synthesized prior to introduction of the reporter. Staf is a zinc finger protein of the TFIIIA type (Brown et al., 1985; Miller et al., 1985) and contains an activation domain rich in acidic amino acid residues.
Results Molecular cloning of the cDNA encoding the factor which binds at the tRNAsec activator element The sequence of the activator element (AE) of the X.laevis tRNASec gene (Myslinski et al., 1992) was used to screen 3777
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an X.laevis mature oocyte XZap expression library. Three positive clones, XMl, XM2 and XM3, were identified after screening 400 000 plaques with a multimerized doublestranded oligonucleotide containing the AE sequence. Phagemids Ml, M2 and M3 containing the inserts arising from XM1, XM2 and XM3 were excised in vivo. To examine whether a protein encoded by M1, M2 or M3 specifically binds to the AE sequence, extracts were prepared from transformed bacteria and subjected to gel mobility shift assays and DNase I footprinting. When extracts prepared from bacteria expressing a protein from M2 were incubated with 32P-labeled AE and submitted to gel electrophoresis, two major shifted bands were obtained (Figure 1A, lane 2). These could not be obtained from bacteria harboring pBluescript SK only (Figure IA, lane 1). Competition experiments indicated that the retarded bands must be relevant to a specific interaction of the DNA binding protein with the AE sequence, since 500and 1500-fold molar excesses of unlabeled AE competed efficiently with the labeled AE for formation of the complexes giving rise to the shifted bands (Figure IA, lanes 3 and 4). In contrast, the mutant AE had no effect (Figure IA, lanes 5 and 6). In an earlier work we observed that HeLa nuclear extracts yielded one single retarded band in gel shift experiments (Myslinski et al., 1992; Figure 2B, lanes 2 and 3). To determine if the two shifted bands observed in Figure 1A could originate from the binding of monomeric or dimeric forms of the protein, gel shift experiments were designed in which increasing amounts of bacterial extracts were added. No differences were observed in the intensity of the two bands with respect to the amount of extract (data not shown). This suggests that the origin of the two complexes results from truncation of the protein occurring by proteolytic cleavage in the bacterial extract, but not in HeLa nuclear extracts. DNase I footprint analysis was performed with a DNA fragment lying between -262 and -123 relative to the transcription start site of the X.laevis tRNAsec gene. Figure lB shows that the bacterial extract expressing the DNA binding protein protected a sequence from -215 to -187 on the upper, non-coding strand and -219 to -190 on the lower, coding strand. The protected region contains in its central part the AE sequence. Results presented in Figure IA and B with the M2-encoded protein were also obtained with bacterial extracts expressing Ml and M3 (data not shown). The DNA binding analyses indicate that the cloned cDNA encodes a protein factor that specifically binds the AE sequence of the selenocysteine tRNA gene. We called this protein the selenocysteine tRNA gene transcription activating factor or Staf.
Recombinant and endogenous Staf bind to DNA with similar sequence specificity We next asked whether the bacterially produced protein binds DNA with the same sequence specificity as the DNA binding activity found in HeLa nuclear extracts (Myslinski et al., 1992). The DNA binding properties of these two types of proteins were compared in an electrophoretic mobility shift assay. Figure 2 shows the capacities of five mutant versions of the activator element (depicted in Figure 2A) to bind these two proteins. The 3778
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Fig. 1. Binding specificity of bacterially synthesized recombinant Staf protein. (A) Gel shift assay. Extracts from bacteria not harboring (lane 1) or harboring (lanes 2-6) the M2 phagemid were incubated with 60 fMol 32P-labeled double-stranded oligonucleotide AE. Incubation was in the absence (lane 2) or presence of a 500- to 1500fold molar excess of unlabeled AE (lanes 3 and 4 respectively) or unlabeled mutant AE (AEmut, lanes 5 and 6 respectively). The AEmut sequence contains the substitution S(-209/-195) described in Myslinski et aL (1992). (B) DNase I footprinting analysis. The bacterial extract containing the M2 cDNA product was incubated with a BamHI-Bglll DNA fragment of the tRNAsec gene encompassing 140 bp from -262 to -123 upstream of the coding region. The upper (non-coding strand) or lower (coding strand) were 5'-end-labeled at the BamHl or Bglll sites respectively. Probes submitted to G+A chemical cleavage were used as markers (lanes 1 and 5). Lanes 2 and 6, probes incubated in the absence of proteins (-); lanes 3 and 7, probes incubated with 1 111 bacterial extract; lanes 4 and 8, probes incubated with 2 gl bacterial extract. The protected region is indicated on the right and left sides and diagramed at the bottom of the figure by a solid bar. The AE sequence is boxed.
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Fig. 2. Endogenous and recombinant Staf exhibit identical binding properties. The upper strand sequences of the wild-type and mutant AE used in this study are indicated (A). Band shift analysis was performed using nuclear extracts from HeLa cells (B) or the bacterially expressed recombinant protein (C). A 157 bp end-labeled wild-type fragment or a 137 bp end-labeled fragment containing mutant versions of the AE were used in the binding studies. (B) Lanes 1, 4, 7, 10, 13 and 16, no protein added (-); lanes 2, 5, 8, 11, 14 and 17, 2 gli HeLa nuclear extract; lanes 3, 6, 9, 12, 15 and 18, 4 1t HeLa nuclear extract. The arrowhead indicates the position of the specific complex. (C) Lanes 1, 4, 7, 10, 13 and 16, no protein added (-); lanes 2, 5, 8, 11, 14 and 17, 1 g1 20-fold diluted bacterial extract; lanes 3, 6, 9, 12, 15 and 18, 1 g.l 10-fold diluted bacterial extract.
endogenous (Figure 2B) and recombinant Staf (Figure 2C) bind to a variable extent the four mutant sequences S(-209/-207), S(-206/-201), S(-200/-198) and S(-197/ -195), which partially substitute the AE. In contrast, no binding was observed with the S(-209/-195) mutant, which totally substitutes the AE, either with the endorecombinant Staf. More importantly, the relative affinities for the different mutant DNAs are identical for the endogenous (Figure 2B) and cloned Staf (Figure 2C) and can be correlated with the in vivo transcriptional activity of the tRNASec gene harboring the same mutations in the AE (Myslinski genous or
Staf is a novel DNA binding protein The complete nucleotide sequence of the Staf cDNA is presented in Figure 3A. The 2973 nt long cDNA is a composite sequence obtained from the 2820 nt long M2 cDNA (positions 154-2973) and 153 bp of additional sequence 5' to the M2 clone obtained by PCR on another cDNA library. Isolation and sequencing of a genomic clone containing this additional region revealed that it actually belongs to Staf. The Staf nucleotide sequence ends with a stretch of A residues located 12 nt downstream from an AATAAA polyadenylation consensus sequence. The longest open reading frame extends from nucleotide positions 28-1827 (Figure 3A). It has the potential to encode a novel protein of 600 amino acids, yielding an unmodified molecular weight of 64.9 kDa. The coding sequence is preceded within the cDNA by a 5' flanking region of 27 nt. The presence at position 7 of an in-frame UAG stop codon upstream of the putative initiation codon implies that the coding region does not extend beyond the AUG at position 28. The coding region is followed by a long 3' untranslated region comprising 1128 nt containing stop codons in all reading frames. This 3' untranslated region contains no AUUUA elements which may act to decrease the mRNA half-life (Caput et al., 1986). Southern blot analysis of genomic X.laevis DNA revealed that the gene coding for Staf is single copy (Figure 4A). Northern blot analysis indicated the existence in X.laevis ovary of a single mRNA of -3 kb specific for Staf (Figure 4B). Seven tandemly repeated zinc fingers contain the DNA binding domain of Staf Staf encodes seven tandemly repeated zinc fingers of the C2-H2 type (Brown et al., 1985; Miller et al., 1985), located between amino acid positions 267 and 468 (Figure 3A). Figure 3B displays the sequence alignment of the seven repeats. One can observe that the first six are of the C-X4-C-X3-F/Y-X5-L-X2-H-X3-H type (X stands for any amino acid) except that the leucine (L) residue is not found in the fourth and fifth fingers, where it is replaced by an arginine (R) and a tyrosine (Y) respectively. The seventh, however, is of the C-X2-C-X3-Y-X5-L-X2-H-X4-H type. The sequence linking the last histidine residue of one zinc finger to the first cysteine of the next is highly conserved, giving rise to the consensus TGEK/RPYX. Part of this sequence is also observed preceding the first zinc finger, between positions 260 and 263 (Figure 3A). To determine whether the zinc finger repeat itself is able to bind the tRNAs5 gene activator element, a recombinant fragment of Staf corresponding to residues 249-475 was expressed in bacteria and used for footprinting experiments. The footprint obtained (data not shown) is identical to that obtained with the entire Staf protein (Figure 1B). These results indicate that a part of or the total zinc finger repeat constitutes the DNA binding domain of Staf. Structural features of Staf In addition to the zinc fingers, analysis of the protein sequence revealed several additional features. Examination 3779
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