DNA-Binding Domain - NCBI

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Oct 21, 1986 - lated from free DNA after visualization of the bands on .... their positions relative to the transcriptional start sites are variable, with no apparent ...
Vol. 7, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1987, p. 1256-1266 0270-7306/87/031256-11$02.00/0 Copyright © 1987, American Society for Microbiology

Expression of qa-lF Activator Protein: Identification of Upstream Binding Sites in the qa Gene Cluster and Localization of the DNA-Binding Domain JAMES A. BAUM,* ROBERT GEEVER, AND NORMAN H. GILES Department of Genetics, University of Georgia, Athens, Georgia 30602 Received 21 October 1986/Accepted 2 December 1986

The qa-iF regulatory gene of Neurospora crassa encodes an activator protein required for quinic acid induction of transcription in the qa gene cluster. This activator protein was expressed in insect cell culture with a baculovirus expression vector. The activator binds to 13 sites in the gene cluster that are characterized by a conserved 16-base-pair sequence of partial dyad symmetry. One site is located between the divergently transcribed qa-iF and qa-IS regulatory genes, corroborating prior evidence that qa-iF is autoregulated and controls expression of the qa-iS repressor. Multiple upstream sites located at variable positions 5' to the qa structural genes appear to allow for greater transcriptional control by qa-IF. Full-length and truncated activator peptides were synthesized in vitro, and the DNA-binding domain was localized to the first 183 amino acids. A 28-amino acid sequence within this region shows striking homology to N-terminal sequences from other lower-eucaryotic activator proteins. A q4-1F(Ts) mutation is located within this putative DNA-binding domain.

The qa gene cluster comprises a contiguous set of genes that control quinate-shikimate utilization in the filamentous fungus Neurospora crassa. The 17.3-kilobase gene cluster contains five structural genes and two regulatory genes tightly linked to the centromere of linkage group VII (Fig. 1) (14). The entire gene cluster has been sequenced, and the 5' and 3' ends of the major mRNA transcripts have been determined (1, 14, 20, 43). Transcription of the qa genes is induced 50- to 1,000-fold by quinic acid and is coordinately regulated by qa-JF and qa-JS. These two genes are constitutively transcribed at low levels but are also subject to induction by quinic acid and to autoregulation (14, 20, 33, 43). All qa genes require the protein encoded by the qa-JF positive regulatory gene for induction by quinic acid (20, 33). The action of the qa-1F activator protein is inhibited either directly or indirectly by the product of the qa-JS regulatory gene. Genetic analysis of mRNA transcription and chromatin structure in the qa gene cluster has provided evidence that the activator protein interacts at specific sites in the 5'-flanking regions of the qa genes (3, 4, 11, 12). As a result of these studies, a conserved 16-base-pair (bp) sequence element present one or more times 5' to each of the qa genes was identified as a potential binding site for the activator protein. Attempts to isolate the qa-1F activator protein by expression of qa-JF in Escherichia coli have been unsuccessful (A. Easton and J. Baum, unpublished data). Furthermore, expression of the intact qa-JF gene in Saccharomyces cerevisiae with the Adcl expression vector AAH5 (2) appeared to be lethal to the recipient since no transformants could be obtained (J. Baum, unpublished data). As an alternative approach to obtaining high-level expression of this gene, we have employed a baculovirus expression vector capable of directing the expression of foreign genes in lepidopteran cell culture (29). A number of proteins including beta interferon (39), beta-galactosidase (34), and c-myc protein (31) have been expressed at high levels in infected Spodoptera *

frugiperda (fall armyworm) cells with a modified form of the Autographa californica nuclear polyhedrosis virus (AcNPV) in which the foreign gene of interest was inserted behind the viral promoter for the polyhedrin gene. Two features of the baculovirus system make it a useful expression vector system. First, the polyhedrin promoter is exceptionally strong insofar as polyhedrin mRNA and protein accumulate to ca. 25% of total levels within the cell late in infection (8, 34, 40). Second, transcription from the polyhedrin promoter occurs late in infection, after extracellular (infectious) virus is produced and after cellular and most viral genes have been turned off, thus allowing the selective expression of potentially cytotoxic proteins (8). In this paper, we report the overexpression of the qa-JF gene using this novel expression vector system as well as the synthesis of full-length and truncated qa-1F activator peptides in an in vitro transcription-translation system. The results of DNA binding and footprinting experiments with partially purified activator protein are described. In addition, we report the localization of the DNA-binding domain to the amino terminus of the activator protein. MATERIALS AND METHODS and viral DNA. The qa-JF+ gene used in this Plasmids study was originally obtained from wild-type N. crassa 74-OR23-1A (74A) (36). A plasmid subclone of this gene (kindly provided by Alan Easton) in which all but eight nucleotides of the 5' leader sequence had been removed by Bal 31 digestion and a unique BamHI site created (GGATC CCAACATG) was used to isolate a 2.9-kilobase BamHIHpaI restriction fragment containing the intact qa-JF coding region. This fragment was inserted into a polylinker sequence downstream of the baculovirus polyhedrin promoter on the transplacement vector pEV/55 (29) (generously provided by David Miller of the Genetics Institute). The resultant plasmid, pF/55, was propagated in E. coli JM101. AcNPV (strain L-1) DNA was prepared from virus-infected cultures as described by Miller et al. (29) except that virus particles in the clarified medium were pelleted at 25,000 rpm

Corresponding author. 1256

qa-lF ACTIVATOR PROTEIN OF NEUROSPORA CRASSA

VOL. 7. 1987

qa-x

qa-2 qa-3

qa-4 qa-y

qa-1 S

qa-1 F

FIG. 1. Gene organization in the qa gene cluster. Five structural and two regulatory genes (qa-JF and qa-lS) form the 17.3kilobase (kb) qa gene cluster. Arrows represent the major mRNA

genes

transcripts for each

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Growth and infection of insect cells. S. frugiperda IPLBSF21 (fall armyworm) cells (Sf cells) (44) were grown in BML-TC-10 medium (9) (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (GIBCO), 100 units of penicillin per ml, 100 pLg of streptomycin per ml, 0.25 jig of amphotericin B (Sigma Chemical Co., St. Louis, Mo.) per ml and 100 pug of kanamycin (GIBCO) per ml. Cells were maintained in T flasks at 25 to 27°C and passaged every 4 days. Infection of cell monolayers

and suspension cultures, preparation of virus stocks, and plaque assays were performed essentially as described by Miller et al. (29). Construction of recombinant virus. Transfection of Sf cells done by a calcium phosphate coprecipitation method (35). To generate recombinant virus containing the was

polyhedrin promoter-qa-lF gene fusion, Sf cells were seeded a density of 2 x 106 cells per dish, cotransfected with wild-type baculovirus (AcNPV) DNA and pF/55 at a molar ratio of 1:10, and overlaid with 5 ml of fresh medium for 4 to 5 days. Recombinant viruses, not producing polyhedral occlusion bodies owing to the deletion of the native polyhedrin gene, were isolated by plaque assay of the culture medium (34, 38). The presence of the polyhedrin-qa-JF fusion in the recombinants was verified by Southern blot analysis of purified virus DNA (41). Expression of qa-lF in Sf cells. Expression of the qa-1F activator protein in infected Sf cells was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (23) of whole-cell extracts as described by Miller et al. (30) except that cells were seeded at a density of 106 cells per 35-mm dish before infection with virus and onto 60-mm dishes at

labeling with [35S]methionine. Preparation of activator protein extracts. Monolayers of Sf cells (107) on 100-mm dishes were infected with recombinant virus at a multiplicity of infection of 20. At 48 h postinfection, cells were harvested and pelleted at 750 x g for 10 min in an HS-4 rotor. Cells (6 x 107 to 1 x 108) were washed twice in phosphate-buffered saline (35), suspended in 10 ml of extraction buffer (25 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-KOH [pH 7.5], 100 mM KCI, 20% glycerol, 0.2% Triton X-100, 5 mM MgC12, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 40 puM pepstatin A, 20 p.M leupeptin) and disrupted with a hand homogenizer (20 strokes) on ice. The homogenate was centrifuged at 12,000 x g for 20 min in an SS-34 rotor. The resultant pellet was resuspended in extraction buffer (4 to 5 ml), and both the supernatant and pellet fractions were dialyzed twice for 3 h at 4°C against 500 ml of buffer containing 25 mM HEPESKOH (pH 7.5), 100 mM potassium acetate, 10% glycerol, 5 mM magnesium acetate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 p.M

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pepstatin A, and 5 puM leupeptin. The majority of the DNA-binding activity associated with the activator protein (see below) was recovered in the supernatant fraction. As a further purification step, the dialysate was loaded onto a phosphocellulose column (Whatman P-11) equilibrated with column buffer (same as dialysis buffer except that potassium acetate and magnesium acetate were replaced with KCl and MgCl2), and after extensive washing, activator protein was eluted with column buffer containing 600 mM KCl. Eluted fractions containing DNA-binding activity were pooled, concentrated by ultrafiltration, and dialyzed as before. Gel electrophoresis DNA-binding assay. Restriction fragments were end labeled with [y-32P]ATP and polynucleotide kinase by standard procedures (25), and 2 to 10 fmol of each fragment (5,000 to 10,000 Cerenkov cpm) was incubated with activator protein extracts in 25 mM HEPES-KOH (pH 7.5)-S50 mM KCI-5 mM MgCl,-1 mM EDTA-50 ,ug of calf thymus DNA per ml (50-,ul total volume) at room temperature for 20 to 30 min. After this incubation period, 5 ,ul of 50% glycerol-0.05% bromophenol blue was added, and the binding reaction mixtures were then loaded onto a 5% polyacrylamide-10% glycerol gel (acrylamide-bisacrylamide, 39:1 [vol/vol]) and electrophoresed at room temperature. Both Tris phosphate (pH 8.0) and Tris borate (pH 8.3) buffer systems (25) were used in this gel assay without any apparent differences. After electrophoresis, the gel was wrapped in plastic wrap and exposed to XAR-5 X-ray film (Eastman Kodak Co., Rochester, N.Y.) at room temperature. Alternatively, the gel was transferred to a piece of Whatman 3MM paper and dried before autoradiography. DNase I footprinting analysis. Conditions for DNase I footprinting were similar to those used in the DNA-binding assay. For incubations with insect cell extracts, the reaction volume was 100 ,ul. For incubations with in vitro translation extracts, the reaction volume was 20 pul plus 2 pul of translation extract in storage buffer (see below). Incubations with end-labeled fragments (ca. 104 Cerenkov cpm) were for 30 min at room temperature, followed by incubation with DNase I at a final concentratin of 10 p.g/ml for 2 min at room temperature. DNase I reactions with insect cell extracts were terminated by adding 200 p.l of 10 mM Tris hydrochloride (pH 7.6)-20 mM NaCl-20 mM EDTA-50 p.g of calf thymus DNA per ml-0.2% SDS. DNase I reactions with translation extracts were terminated by adding EDTA to 20 mM, and the DNA fragments were subsequently resolved on 4% polyacrylamide gels. Protein-DNA complexes were isolated from free DNA after visualization of the bands on autoradiograms and excising the appropriate areas from the gel. DNAs were electroeluted into dialysis tubing pretreated with 1% Sarkosyl. With both methods, the final steps included extraction with buffered phenol (with 10% m-cresol) and chloroform, precipitation with ethanol, and resolution of the DNA fragments on 8% sequencing gels. In vitro transcription and translation. The DNA template, pSP64/qa-JF, was used for transcription with SP6 polymerase (Promega Biotech, Madison, Wis.) after purification on a CsCl gradient and digestion with the appropriate restriction enzymes. Conditions for transcription were as described by Melton et al. (26) with 1 pug of cleaved template in a 25-pul reaction mixture. RNA products were extracted with phenol-chloroform, precipitated with ethanol, and suspended in 10 mM Tris hydrochloride (pH 7.5)-i mM EDTA. Each sample was divided into equal aliquots for translation in a 50-plI reaction mixture with rabbit reticulocyte extracts and under conditions specified by the manufacturer (Promega Biotech). One aliquot was translated without labeled amino

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BAUM ET AL.

acids, the other with 75 ,uCi of [35S]methionine (specific activity, ca. 1,500 Ci/mmol; Amersham Corp., Arlington Heights, Ill.). Incorporation of [35S]methionine into protein was determined by incubating 1 RI of translation extract with 0.5 ml of 1 M NaOH-1.5% H202 at 37°C for 10 min and then with 2 ml of cold 25% trichloroacetic acid-2% Casamino Acids (Difco Laboratories, Detroit, Mich.) on ice for 30 min. Precipitated protein was collected on glass fiber filters and washed successively with 5 ml of cold 8% trichloroacetic acid and 5 ml of acetone, and counts were measured by liquid scintillation counting with Opti-Fluor (United Technologies Packard). On the average, 10 ng of qa-1F protein was synthesized per 50-pI translation reaction. Translation extracts were stored at -20°C after the addition of an equal volume of 2 x storage buffer (20 mM Tris hydrochloride [pH 7.4], 100 mM KCl, 0.2 mM EDTA, 0.2 mM dithiothreitol, 80% glycerol).

RESULTS Isolation of recombinant virus. Screening for recombinant viruses containing the polyhedrin promoter-qa-JF gene fusion instead of the native polyhedrin gene was facilitated by the fact that such recombinants produce plaques that lack the highly refractive occlusion bodies composed primarily of polyhedrin (34, 38). These occluded virus-minus (OV-) plaques can be distinguished from wild-type (OV+) plaques by using a dissecting microscope equipped with an oblique light source. Cotransfections of Sf monolayers with wildtype baculovirus (AcNPV) DNA and the transplacement vector pF/55 (containing the polyhedrin-qa-JF fusion) yielded viral plaques at frequencies of 500 to 1,000/,ug of viral DNA of which OV- plaques represented 0.1 to 1.0%, within the range previously reported (34, 38). OV- plaques were picked, and the recombinant virus isolates were purified by repeated rounds of plating onto Sf monolayers. DNAs from recombinant virus stocks were screened by restriction enzyme analysis on Southern blots for the presence of the intact qa-JF gene. The results of the restriction enzyme analysis were consistent with the expected gene replacement event. Expression of qa-IF in Sf cells. We prepared extracts from Sf monolayers infected with either recombinant or wild-type virus to determine whether the qa-1F activator protein was expressed in Sf cells. At 0, 48, and 72 h postinfection, 106 cells were washed with phosphate-buffered saline, labeled with 50 puCi of [35S]methionine for 1 h, and extracted directly in 2x SDS sample buffer (23). The time course of protein expression after viral infection was monitored by SDSPAGE followed by staining with Coomassie blue R250 and autoradiography (Fig. 2). Cells infected with wild-type virus (AcNPV) exhibited high levels of polyhedrin synthesis at 48 h postinfection, as indicated by the 33,000-molecular-weight band (P) observed on the autoradiogram (Fig. 2a). Cells infected with recombinant virus (AcNPV+F) lacked polyhedrin synthesis altogether and instead expressed a novel high-molecular-weight protein (F). The adjacent panel (Fig. 2b) shows that this protein is readily detectable by Coomassie blue staining at 48 and 72 h postinfection. The reduction in synthesis of the presumed activator protein at 72 h postinfection parallels the reduction in synthesis of polyhedrin (Fig. 2a) (34). The apparent molecular weight of 100,000 is slightly larger than that inferred for the activator protein from the DNA sequence (88,960) (L. Huiet, Ph.D. thesis, University of Georgia, Athens, 1983). However, the mobility of this protein on SDS-polyacrylamide gels is indis-

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HOURS POST-INFECTION FIG. 2. Overexpression of qa-JF' in Sf cells. Sf monolayers (106 cells) were infected with wild-type (AcNPV) or recombinant (AcNPV+ F) baculovirus at a multiplicity of infection of 20. At 0, 48, and 72 h postinfection, cells were labeled with 50 ,uCi of [35S]methionine for 1 h, extracted directly in SDS sample buffer, and analyzed by SDS-PAGE. (a) Autoradiogram of a 7.5% SDSpolyacrylamide gel; (b) the same gel stained with Coomassie blue R250. Molecular weight standards (lane M): phosphorylase b (97,000 [97K]), bovine serum albumin (68,000), and ovalbumin (43,000). Cells infected with wild-type virus synthesize the 33,000-dalton (33K) polyhedrin protein (P), while cells infected with recombinant virus do not. Instead, cells infected with recombinant virus produce a novel protein (F) of ca. 100,000 daltons, roughly the size expected for the activator protein.

tinguishable from that of activator protein synthesized by in vitro methods (see below), suggesting that the migration on SDS gels is anomalous. qa-IF activator is a DNA-binding protein. Genetic evidence indicated that the qa-1F activator protein was likely to be a DNA-binding protein. Since the migration of protein-DNA complexes on native polyacrylamide gels is different from the migration of free DNA (7, 10), a gel electrophoresis technique was used to measure specific DNA-binding activity in crude extracts of Sf cells superinfected with wild-type or recombinant virus. In this assay, specific DNA binding is detected as a discrete retardation in the electrophoretic mobility of 32P-labeled restriction fragments. Previous analyses had implicated a 104-bp region 5' to the qa-2 gene as a probable target for activator binding (12). End-labeled restriction fragments spanning this region and other 5' regions between the two qa structural genes, qa-x and qa-2 (Fig. 3a), and the qa-lF and qa-lS regulatory genes (Fig. 3b) were incubated with insect cell extracts (see Materials and Methods) and resolved on a 5% polyacrylamide gel. In Fig. 3a, three restriction fragments of 355, 214, and 146 bp (RsaI)

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qa-lF ACTIVATOR PROTEIN OF NEUROSPORA CRASSA

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TABLE 1. Summary of DNase I footprinting analysis and DNA binding assays

Gene

Positiona

Conserved

Binding

sequence' *

qa-2-qa-x qa-2 qa-2 qa4 qa4 qa-3 qa-3 qa-3 qa-y qa-y qa-y qa-lS qa-lF-qa-IS

TCGGCT TTTCTT GGGGAC ACGGCC CCCACC ACTGCT CCCAAA AAAATG CTGGCT TCCCCC GCTGCC CTGATC ACAAAA

-510--512 -391 -127 -510 -374 -449 -264 -70 -681 -512 -412 +236 -144--226

Consensus (13 sequences)

GGATGA GGATAA GGGTAA GGCTAA CGTTAA GGGTAA GGCAAA GGGGAA GGGTAA GGTTAT GGCTCA GGATGA GGATAA

TCGC GTGT TGC T GTA T ACGC TAAC TGGC ACA T ACAC GTGA ACAA

TTAACC TAACCC TTATCC TTAACA TTATTC TTAAGC TCATCC TTATAG TTTTCC TCATCC TCATCA TTCTCC TTATCC

GGRTAA

RYRY

TTATCC

GTT T ATCC

GTTGAT TTTCTT GCTCGT CGCAAG CGCTGA GGTAGG TGAATC CCACGC GTTCAT TCACCC GCTTCA AGACAT TCCCAA

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