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Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA

1997; Springer Nature; Volume: 16; Issue: 13 Linguagem: Inglês

10.1093/emboj/16.13.3974

1460-2075

Adriana Ravagnani, Lisette Gorfinkiel, Tim Langdon, George Diallinas, Élisabeth Adjadj, Stéphane Demais, Diana Gorton, Herbert N. Arst, Claudio Scazzocchio,

Genomics and Phylogenetic Studies

Article1 July 1997free access Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA Adriana Ravagnani Adriana Ravagnani Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK Present address: Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, SY23 3DA UK Search for more papers by this author Lisette Gorfinkiel Lisette Gorfinkiel Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Present address: Seccion Bioquímica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK Search for more papers by this author Timothy Langdon Timothy Langdon Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK Present address: Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed, SY23 3EB UK Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK Search for more papers by this author George Diallinas George Diallinas Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Present address: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, PO Box 1527, Heraklion, 71 110 Crete, Greece Search for more papers by this author Elisabeth Adjadj Elisabeth Adjadj U350 INSERM, Institut Curie, Centre Universitaire, Bâtiment 112, 91405 Orsay, Cedex, France Search for more papers by this author Stéphane Demais Stéphane Demais Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Search for more papers by this author Diana Gorton Diana Gorton Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ Essex, UK Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK Search for more papers by this author Herbert N. Arst Jr Herbert N. Arst Jr Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK Search for more papers by this author Claudio Scazzocchio Claudio Scazzocchio Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ Essex, UK Search for more papers by this author Adriana Ravagnani Adriana Ravagnani Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK Present address: Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, SY23 3DA UK Search for more papers by this author Lisette Gorfinkiel Lisette Gorfinkiel Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Present address: Seccion Bioquímica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK Search for more papers by this author Timothy Langdon Timothy Langdon Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK Present address: Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed, SY23 3EB UK Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK Search for more papers by this author George Diallinas George Diallinas Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Present address: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, PO Box 1527, Heraklion, 71 110 Crete, Greece Search for more papers by this author Elisabeth Adjadj Elisabeth Adjadj U350 INSERM, Institut Curie, Centre Universitaire, Bâtiment 112, 91405 Orsay, Cedex, France Search for more papers by this author Stéphane Demais Stéphane Demais Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Search for more papers by this author Diana Gorton Diana Gorton Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ Essex, UK Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK Search for more papers by this author Herbert N. Arst Jr Herbert N. Arst Jr Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK Search for more papers by this author Claudio Scazzocchio Claudio Scazzocchio Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ Essex, UK Search for more papers by this author Author Information Adriana Ravagnani1,2, Lisette Gorfinkiel3,4,9, Timothy Langdon1,5,9, George Diallinas3,6, Elisabeth Adjadj7, Stéphane Demais3, Diana Gorton8,9, Herbert N. Arst1 and Claudio Scazzocchio3,8 1Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Ducane Road, London, W12 0NN UK 2Present address: Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, SY23 3DA UK 3Institut de Génétique et Microbiologie, Unité de Recherche Associé au CNRS, 1354, Bâtiment 409, Université Paris-Sud, Centre d'Orsay, France 4Present address: Seccion Bioquímica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay 5Present address: Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed, SY23 3EB UK 6Present address: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, PO Box 1527, Heraklion, 71 110 Crete, Greece 7U350 INSERM, Institut Curie, Centre Universitaire, Bâtiment 112, 91405 Orsay, Cedex, France 8Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ Essex, UK 9Present address: Enfield and Haringey Health Authority, Alexandra Place, Lower Park Road, London, N11 1ST UK ‡L.Gorfinkiel and T.Langdon contributed equally to this work The EMBO Journal (1997)16:3974-3986https://doi.org/10.1093/emboj/16.13.3974 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A change of a universally conserved leucine to valine in the DNA-binding domain of the GATA factor AreA results in inability to activate some AreA-dependent promoters, including that of the uapA gene encoding a specific urate–xanthine permease. Some other AreA- dependent promoters become able to function more efficiently than in the wild-type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild-type and mutant forms of AreA. In vitro binding studies of wild-type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild-type and mutant AreA–DNA complexes derived from the atomic coordinates of the GATA-1–AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA-binding sites, is sufficient to ensure activation of transcription of the uapA gene. Introduction The areA gene encodes a factor necessary for the efficient transcription of >100 genes involved in the utilization of nitrogen sources in Aspergillus nidulans (Arst and Cove, 1973). An isofunctional, homologous gene, nit-2, has been described in Neurospora crassa (Fu and Marzluf, 1990), while the GLN3, DAL80, NIL1 and NIL2 genes regulate in a less straightforward fashion the utilization of some nitrogen sources in Saccharomyces cerevisiæ (Cunningham and Cooper, 1991; Minehart and Magasanik, 1991; Coffman et al., 1995; Stanbrough et al., 1995; D.Rowen, N.Esiobu and B.Magasanik, personal communication). The single DNA-binding domains of these fungal proteins show striking similarities to the zinc fingers and adjacent downstream basic regions of the metazoan GATA factors (Kudla et al., 1990), more particularly to the carboxy-terminus of the two DNA-binding domains. The AreA protein mediates nitrogen metabolite repression by responding to the glutamine concentration in the cell. Glutamine is thought to destabilize the areA mRNA through an unknown mechanism, and to prevent, via its interaction with another protein, the binding of AreA to its cognate DNA sites (Platt et al., 1996a). Even within the same pathway, different promoters show varying degrees of sensitivity to nitrogen metabolite repression. Thus, in the purine degradation pathway, the promoters of the permeases coded by the uapA and uapC genes are exquisitively sensitive to nitrogen metabolite repression, while the expression of the hxA and uaZ genes is never repressed completely (Oestreicher and Scazzocchio, 1995). Two mutations that alter differentially the expression of a number of genes controlled by AreA result in substitutions of a universally conserved leucine in the seventh position of the Zn finger loop (Leu683) by valine and methionine respectively (Kudla et al., 1990). Strains carrying the valine substitution (areA102) show, inter alia, a drastically increased expression of the amdS gene, encoding acetamidase, and almost null expression of the genes coding for the specific uric acid–xanthine permease and for the broad specificity purine permease (uapA and uapC respectively, Arst and Scazzocchio, 1975; Hynes, 1975; Scazzocchio and Arst, 1978; Gorfinkiel et al., 1993; Diallinas et al., 1995). Strains carrying the methionine substitution (areA30, areA31) have a phenotype which is qualitatively a less extreme mirror image of that of strains carrying the valine substitution (Arst and Scazzocchio, 1975; Gorton, 1983). Strains carrying the methionine substitution have been isolated as revertants of areA102 on uric acid as nitrogen source (see below, Arst and Scazzocchio, 1975) A strain with an identical phenotype, isolated in another laboratory (areA200, Hynes, 1975), was shown to carry an identical substitution (T.Langdon and H.N.Arst,Jr, unpublished data). As a consequence of the total loss of uric acid uptake activity, areA102 strains cannot utilize uric acid as nitrogen source. We have shown that it is possible to obtain revertants which grow on uric acid. The cis-acting mutations responsible for the suppression of areA102 specifically for growth on uric acid are adjacent to either the uapA or the uapC genes (Arst and Scazzocchio, 1975; Gorfinkiel et al., 1993; Diallinas et al., 1995). We speculated that such revertants may have acquired AreA-binding sites similar to those present in genes which are expressed normally or are overexpressed in the areA102 strains. One such suppressor, uapA100 (previously called uap100), was shown to affect drastically the expression of the uapA gene. In an areA+ background, uapA100 results in constitutivity and overexpression of the uapA gene. However, this mutation does not suppress a total loss-of-function areA allele, nor a null mutation in uaY, the gene coding for the transcription factor mediating specific induction by uric acid. uapA100 is a 164 bp duplication. This duplication includes the single specific UaY-binding site (Suárez et al., 1995) and a couple of putative, almost adjacent, AreA-binding sites, the sequence being CGATAGagAGATAA. Data from a number of laboratories have shown that while AreA and other GATA factors are able to bind to single WGATAR sites (Merika and Orkin, 1993; Ko and Engels, 1993; D.Gómez, B.Cubero and C.Scazzocchio, unpublished data; A.Ravagnani and H.N.Arst,Jr, unpublished data), physiologically relevant sites occur often in direct or inverted repeats separated by a few base pairs (Martin et al., 1989; Strauss et al., 1992; Feng et al., 1993; Langdon et al., 1995; M.I.Muro, J.Strauss, and C.Scazzocchio, unpublished data). It was shown in vitro that low affinity or even WGATTR sites can be bound if nearby there is a strong binding site. This cooperativity is evident even in fusion proteins containing little more than the DNA-binding domain (Feng et al., 1993; D.Gómez, B.Cubero and C.Scazzocchio, unpublished data). Although the consensus for GATA factors is usually written WGATAR, CGATAR sites are recovered at low frequency in random selection experiments (Ko and Engel, 1993; Merika and Orkin, 1993). A physiological function for such CGATAR sites has not been described to date. We speculated that the non-canonical cytosine present in the CGATAGagAGATAA sequence of the uapA promoter is indeed the base which results in the inability of the uapA promoter to respond to an AreA102 mutated protein. We thus analysed a number of mutations which were obtained as specific revertants on uric acid but that showed a rather different phenotype from uapA100. These mutations accommodate the AreA102 (AreAVal) protein without affecting the non-induced levels of uapA expression. The NMR solution structure of the GATA-1–DNA complex showed that the conserved leucine in position 7 of the loop of the Zn finger makes a number of hydrophobic contacts, including one with the first base of the AGATAA sequence used in NMR studies (Omichinski et al., 1993). We had speculated that the gene-specific effects of the mutations at position 683 might be due to the loss or strengthening of these hydrophobic interactions (Scazzocchio, 1990). Results Selection and genetic characterization of specific suppressors of areA102 We previously have described promoter mutations which suppress the areA102 mutation specifically for growth on uric acid and xanthine. A number of these are adjacent to the uapC locus (Diallinas et al., 1995), but only one, a 164 bp duplication, is a cis-acting mutation upstream of the coding region of the uapA gene (Arst and Scazzocchio, 1975; Gorfinkiel et al., 1993). We sought to isolate additional suppressors mapping in the promoter of uapA. Spores from a biA1 areA102 strain were plated on medium containing uric acid as sole nitrogen source. Strains able to grow on uric acid were selected either as spontaneous mutants or after UV mutagenesis (15% survival). Wild-type revertants or those able to grow on uric acid because the Val683 has been substituted by a methionine can be distinguished easily from suppressors specific for utilization of uric acid and presumably mapping in the uapA or uapC promoters by their effects on growth on a number of unrelated nitrogen sources (Arst and Scazzocchio, 1975; Kudla et al., 1990). One spontaneous and seven UV-induced mutant strains carrying uric acid–xanthine specific suppressor mutations were found. Extragenic, specific suppression was shown for these eight strains by recovery, in every case, of 25% areA102 non-suppressed progeny in crosses with areA+ strains. These eight strains were then crossed to uapA100 areA102 and uapC201 areA102 strains carrying previously characterized uapA and uapC promoter mutations respectively. At least 300 progeny were analysed for each cross. Four strains each carry a suppressor mutation unlinked either to uapA or uapC and were not analysed further. Two strains carry suppressor mutations (designated uapC305 and uapC307) in or very near the uapC promoter. These will be described in a separate publication. Three strains carry suppressors of areA102 which map in or very near the uapA promoter: they are designated uapA302, uapA310 and uapA349. In particular, the phenotypically identical mutations uapA302 and uapA310 did not yield any uapA+ recombinants in, respectively, 1110 and 2680 progeny in crosses in repulsion to uapA100. Mitotic haploidization analysis showed the uapA349 strain to carry a I–VII chromosome translocation. uapA maps to chromosome I (Arst, 1988), and thus this translocation might be identical to the suppressor mutation. The extent of suppression afforded by uapA310 is shown in Figure 1. Figure 1.The specific suppression of the areA102 mutation by different mutations in the HGATAR sites in the uapA promoter is shown. The Petri dishes contain A.nidulans minimal medium with 5 mM ammonium (+) tartrate (NH4+), 700 μM hypoxanthine (Hx) or 700 μM uric acid (UA) as sole nitrogen sources. The genotype of each strain is indicated. areA+ is the wild-type allele (AreALeu), areA600 is a total loss of function, chain termination mutation (Kudla et al., 1990) and is included as a control, areA102 codes for the AreA(Val) protein, uapA+ is the wild-type promoter (CGATAGagAGTATA). uapA100 is a 164 bp duplication within the uapA promoter containing the CGATAGagAGTATA sequence described previously (Gorfinkiel et al., 1993). uapA310 carries the TGATAGagAGATAA pair of sites, uapA500 the CGATAGagTGATAA pair and uapA501 the TGATAAagTGATAA pair of sites (see text). Mutant bases are in bold. Mutation uapA501 contains, in addition to the substitutions in the first base of the HGATAR sites, an inversion between nucleotides 452 and 802 of the uapA promoter (numbering as in Gorfinkiel et al., 1993). This has presumably occurred because a very similar 9 bp sequence flanks these nucleotides. Despite several attempts, it was not possible to obtain a transformant which did not also carry this inversion. Note that this inversion inverts the relative orientation of AreA- and UaY-binding sites in relation to the uapA transcription startpoint. Download figure Download PowerPoint Molecular characterization of the uapA promoter suppressors of areA102 The phenotypically identical mutations uapA302 and uapA310 consist of a single base pair change in the uapA promoter: CGATAGagAGATAA is mutated to TGATAGagAGATAA (positions from nucleotide 653 to 666 in Gorfinkiel et al., 1993, relevant bases in bold). For uapA302, we have sequenced 874 bp upstream of the uapA initiation codon. For uapA310, 160 bp comprising the AreA- and UaY-binding sites were sequenced. It was shown by transformation of a suitable areA102 strain with the 160 bp mutated fragment that the mutation found was sufficient to suppress the areA102 phenotype on uric acid and that the degree of suppression was identical to that found in the original uapA302 and uapA310 stains (Figure 1). No change was detected in the cognate region of uapA349. Southern blots hybridized with a number of probes upstream from the uapA gene showed a translocation breakpoint ∼3.2 kb from the transcription start point (results not shown). This translocation is presumably associated with the weak suppression of areA102 in strains carrying uapA349. As this work concerns specifically the GATA-binding sites, this translocation was not analysed further. Levels of uapA transcripts in strains carrying point mutations in the uapA promoter These are shown in Figure 2. The weak suppression of areA102 by the two identical point mutations is clear, and is similar to the degree of suppression conferred by the uapA100 duplication (Figure 5 of Gorfinkiel et al., 1993). However, while the duplication results in constitutivity and considerably increased expression in an areA+ background (Arst and Scazzocchio, 1975; Gorfinkiel et al., 1993), uapA310 does not result in constitutivity and, in fact, somewhat decreases uapA-induced expression in an areA+ [AreA(Leu)] background. Figure 2.Northern blots showing the steady-state levels of uapA mRNA in areA+, areA102 and suppressor mutations located in the uapA promoter as described in the text. In the right hand panel, the steady-state levels for a uapA310 single mutant are shown. All procedures were carried out in parallel and the RNAs run in the same gel. NI, mycelia grown under non-inducing conditions, I mycelia grown under induced conditions as described in Materials and methods. Download figure Download PowerPoint In vitro binding of wild-type and mutant AreA fusion proteins to the uapA promoter The binding of a His fusion protein containing the AreA DNA-binding domain (residues 468–729) to the relevant region of the uapA promoter was investigated. Figure 3 (top panel) shows that the AreA(Leu) protein binds specifically to a 162 bp probe derived from the uapA promoter and containing the CGATAGagAGATAA sequence. A mutant probe in which both sites have been mutagenized to CGGGAGagCGGGAA (substituted bases in bold) does not bind to AreA(Leu), nor is it able to compete with the binding of a wild-type probe. Figure 3 (bottom panel) shows that the wild-type probe described above has less affinity for the AreA(Val) protein than for the wild-type AreA(Leu) or AreA(Met) proteins. Figure 3.(A) Specificity of binding of the AreA(Leu) protein. Four hundred ng of a His-AreA(Leu) protein were incubated with a 162 bp uapA promoter probe, prepared by PCR following the standard conditions indicated in Materials and methods and comprising the CGATAGagAGATAA sites, or with the same probe mutated in the cognate sites (mutated to CGGGAGagCGGGAA as described in Platt et al., 1996b, see Materials and methods; mutagenized bases in bold). The presence and absence of the His-AreA protein is indicated as '−' and '+' respectively. Competition was with 100 times excess of cold probe. WT designates the wild-type and Mut the mutant probe. (B) Relative affinities of the AreA(Leu), AreA(Val) and AreA(Met) protein for the 162 bp uapA promoter region containing the 5′CGATAGagAGATAA sequence. '−' indicates probe incubated without protein, 'Leu', 'Val' and 'Met' the three different His-AreA fusion proteins used. Increasing quantities of proteins are indicated at the bottom of the figure. Download figure Download PowerPoint Figure 4 (left panel) shows DNase I footprints of the non-template strands of the wild-type uapA promoter, a promoter carrying the uapA310/uapA302 C→T substitution in the first base of the (upstream) CGATAG site and a promoter carrying an A→T mutation in the first base of the (downstream) AGATAA site. The striking result is the considerable loss of protection of the upstream site seen with AreA(Val) protein in the CGATAGagAGATAA promoter. The protection is recovered using both the TGATAGagAGATAA and the CGATAGagTGATAA probes (mutant bases in bold type). Note that improved binding of the AreA(Val) protein to the downstream site of the latter probe restores protection to the pattern observed with the AreA(Leu) protein. This is consistent with a cooperative effect between the AreA molecules occupying the two sites. Interestingly, both mutant probes show additional protected bases with the AreA(Val) mutant. The first bases of the wild-type probe (the G opposite the C in the first position of the upstream site and the T opposite the A in the first position of the downstream site) are not protected by the AreA(Leu), AreA(Val) or AreA(Met) proteins. The A opposite the first T of the upstream site in the TGATAGagAGATAA mutant probe and the A opposite the T in the downstream site of the CGATAGagTGATAA mutant probe (relevant bases in bold type) show clearly increased protection by the AreA(Val) protein. This implies that the AreA(Val) protein binds to TGATAR sites more tightly than the AreA(Leu) or AreA(Met) proteins and/or binds in a conformation resulting in extended protection. The footprint of the template strand (Figure 4, right panel) shows a slight but uniform decrease in protection of the CGATAGagAGATAA and TGATAGagAGATAA probes by the AreA(Val) protein when compared with that afforded by the AreA(Leu) and AreA(Met) proteins. For the latter probe, the T in the fourth position of the downstream site is less protected by the AreA(Val) protein than by AreA(Leu) and AreA(Met). For the CGATAGagTGATAA probe, the G in the second position of the downstream site is sensitive to DNase I in the presence of the AreA(Leu) and AreA(Met) proteins but is clearly protected by the AreA(Val) protein. These subtle changes are consistent with conformational differences between the different AreA–DNA complexes. Figure 4.DNase I protection of the 162 bp uapA promoter sequence containing two HGATAR sites. 'Probe' indicates whether the sites were CGATAGagAGATAA (wild-type promoter), TGATAGagAGATAA (uapA302 and uapA310 mutations) or CGATAGagTGATAA (uapA500 mutation, see below). Only the first bases of the HGATAR sites, here indicated in bold, are shown. 'G ladders' and 'T ladders' show the cognate Maxam and Gilbert (1977) reaction for each of the probes respectively from left to right. DNase I digestions of each probe, in the absence of any protein and in the presence of AreA(Leu), AreA(Val) and AreA(Met), are indicated by −, L, V and M respectively. (A) Non-template strand. The sequence on the side is the protected sequence (complement of CGATAGagAGATAAgc). The arrows indicate the positions of the mutations, visible as the absence of a band, respectively in the G and T ladders. The stars at the left of weak or missing bands indicate the extended protection conferred by AreA(Val) to the mutant probes as discussed in the text. (B) Template strand. The protected sequence, including the CGATAGagAGATAA sequence, is shown on the side. The arrows indicate the mutations, visible in this strand as additional Ts. The stars to the right of bands indicate the specific loss of protection as described in the text. Download figure Download PowerPoint Figure 5.dUTP interference of a uapA promoter probe containing a TGATAGagAGATAA mutant sequence. Leu, Val and Met designate respectively the amino acid present at the seventh position of the Zn finger loop. Differential interference is seen for the T in the first position of the upstream site (see text), while interference for the internal Ts is seen with all three proteins. Note that this gel also shows binding to a non-canonical CGATCG site 27 bp downstream from the two canonical sites. A clear, albeit partial interference is seen for all proteins, for the T in position 4 of the site. This binding and the interference of internal Ts of the canonical GATA sites constitute suitable internal controls for this experiment. Download figure Download PowerPoint The effect of a T in the first position of an HGATAR site (where H stands for A, T or C) is shown clearly by the dUTP interference experiment of Figure 5. In this method, random substitution of thymines by uracil residues is used to investigate the role of a methyl group in position 5 of the pyrimidine ring in a given protein–DNA interaction (Pu and Struhl, 1992). A striking interference is seen for the TGATAGagAGATAA probe with the AreA(Val) protein. Thus, a 5-methyl group in the first base is only essential when a valine is the amino acid in position 7 of the Zn finger loop. Moreover, there is clear preferential binding with the uracil-substituted probe for the AreA(Leu) and AreA(Met) proteins (comparing the lanes corresponding to the bound and unbound probes). These results show clearly that a methyl group on carbon 5 facilitates the binding of AreA(Val) while it interferes with the binding of AreA(Leu) and perhaps, even more strongly, AreA(Met). Interaction between the hydrophobic amino acid in position 7 and the first base pair of an HGATAR sequence The results obtained by changing systematically the first base of the CGATAG sequence in two different contexts are shown in Figure 6. In one context, the downstream binding site is the AGATAA wild-type sequence, in the other this sequence was mutated to TGATAA. We have investigated qualitatively, in conditions of excess probe, the binding of AreA(Leu), AreA(Val) and AreA(Met) to these different probes. To investigate the importance of a 5-methyl (pyrimidine) group in the first position of the upstream site, we have constructed, in addition to probes with thymine at this position, also probes with 5-methylcytosine. Figure 6.Systematic variation of the first base of the upstream GATA site (5′CGATAG) of the uapA promoter. In all experiments, 52 bp probes, derived from the wild-type promoter as described in Materials and methods, were used. The probes were all prepared using a standard protocol and thus are in approximately equimolecular amounts and in excess for the binding reaction with all proteins. Mutations were carried out in (A) wild-type (AGATAA) downstream site context and (B) in a mutant (TGATAA) downstream site context. The bases mutated in one or more of the different probes are shown in bold. CM indicates 5-methylcytosine Each group of four tracks corresponds to a given probe, '–' indicates absence of protein and L, V a