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Deletion Mapping a Eukaryotic Promoter Proc. NatL Acad. Sci. USA Vol. 78, No. 7, pp. 4461-4465, July 1981 Genetics Deletion mapping a eukaryotic promoter (gene expression/gene regulation/yeast his3 gene/transcription/recombinant DNA) KEVIN STRUHL Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, England CB2 2QH Communicated by Sydney Brenner, March 30, 1981 ABSTRACT The phenotypes of 24 mutants that successively lication (9, 10). Third, because only a small number ofdeletion delete DNA sequences adjacent to the 5' end ofthe Saccharomyces mutations have been tested, it is unclear whether their phe- cerevisiae (yeast) his3 structural gene are described. Deletions notypes are due to the absence of a particular DNA sequence retaining >155 base pairs before the mRNA coding sequences are or to effects ofthe fused sequences at and around the noveljoint. phenotypically indistinguishable from the wild-type his3 allele. Thus, to analyze a promoter, it is important to obtain a set of Deletions having end points between 113 and 65 base pairs before derivatives that can be related to each other easily and to use the transcription initiation site express his3 at reduced levels. a simple experimental system that mimics Mutations retaining <45 base pairs are indistinguishable from null physiological alleles ofthe his3 locus. These results indicate (i) that a sequence(s) conditions. located 113-155 base pairs upstream from the transcribed region The experiments discussed here were designed to determine is necessary for wild-type expression and (ii) that the T-A-T-A box the minimum contiguous DNA sequence necessary for a func- (a sequence in front of most eukaryotic genes) is not sufficient for tional eukaryotic promoter. A series of24 deletion mutants that wild-type promoter function. Thus, the yeast his3 promoter region successively remove DNA sequences adjacent to the 5' end of appears large when compared with prokaryotic promoters, sug- the mRNA coding region of the Saccharomyces cerevisiae gesting that it may be more complex than a simple site of inter- (yeast) his3 gene are described. The deletion mutant DNAs are action between RNA polymerase and DNA. introduced back into yeast cells. In some of the experiments, the transformed yeast cells contain one to three copies of the A promoter is the genetic element necessary for maximal po- transforming DNA replicating autonomously. In others, the tential expression of a particular structural gene. It is distin- cells contain one copy of the transforming DNA integrated at guished from the structural gene itself and from elements that the normal chromosomal location for his3. Thus, the deletion regulate the expression of the gene (1). Experimentally, it is mutant DNAs are analyzed in their native physiological defined by cis-dominant mutations closely linked to the struc- environment. tural gene that alter the basal level ofexpression independently of gene regulation (2). Escherichia coli promoters have been MATERIALS AND METHODS characterized molecularly by comparing the structures and functions ofwild-type and promoter-mutant DNAs both in vivo The eight deletions described previously all derive from Agt4- and in vitro (for review, see ref. 3). In vitro systems using pu- Sc2601, a hybrid containing the intact his3 gene flanked by 2.5 rified E. coli RNA polymerase and purified DNA templates and 6.9 kilobases (kb) ofyeast DNA (see Figs. 1 and 2) (14). Most mimic the phenotypes in vivo of wild-type and mutant genes were formed by an in vivo "illegitimate" recombination event (4, 5). The base pairs altered in many promoter mutants are mediated by the bacteriophage A int gene; these have one com- those that interact specifically with RNA polymerase (6, 7). mon deletion end point at the A attachment site (att). This prop- Thus, the general view is that E. coli promoters are regions of erty not only permits accurate physical mapping by simple re- DNA that specifically bind RNA polymerase such that tran- striction endonuclease cleavage but also results in the fusion of scription of the structural gene is correctly initiated. the identical A sequence to different points in the his3 gene What is the molecular nature of a eukaryotic promoter? Is (14). Unfortunately, his3 end points ofthese int-mediated dele- it simply an RNA polymerase binding site analogous to a pro- tion mutants are not located randomly because the recombi- karyotic promoter? Or is it a more complex structure that in- nation event depends on partial sequence homology between volves specific interactions with histones and nonhistone pro- att and yeast DNA (8). Therefore, I developed a new vector teins found associated in chromatin? (Agt9) that retains the possibility ofobtaining int-mediated dele- These questions have been approached by cloning individual tions and also prevents re-isolation of those described already. intact eukaryotic structural genes, isolating and physically char- Agt9-Sc2601 (Fig. 1D) was constructed by ligating the ap- acterizing mutated derivatives, and assaying their "pheno- propriate partial EcoRI cleavage products of Agt-AC' (Fig. 1B; types. " The phenotypes ofcloned DNAs have been determined ref. 15) and Agt4-Sc2601 (Fig. 1C; ref. 14). The key feature of by transformation back into the native organism (8-10), by this phage is that int and att are inverted with respect to his3. microinjection into frog oocytes (11), and by transcription in Thus, the partial sequence homology favoring int-mediated vitro (12, 13). Although derivatives behaving in altered ways deletion formation will occur between the inverted att and the have been obtained, interpretation of these experiments is not his3 sequences. In fact, hot spots for deletions of Agt9-Sc2601 simple. First, in the microinjection and in vitro assay systems, predicted from the his3 sequence (8) should occur at a number the phenotypes are determined under nonphysiological con- of sites between 44 and 300 base pairs (bp) upstream from the ditions. Second, it has been difficult to distinguish effects on transcribed region. gene expression from those on gene regulation and DNA rep- Deletion mutations that have end points within this region were isolated as follows. Thirty-six independent stocks ofAgt9- The publication costs ofthis articlewere defrayed in part by page charge Sc2601 were treated with EDTA to kill selectively the parent payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: kb, kilobase(s); bp, base pair(s). 4461 4462 Genetics: Struhl Proc. NatL Acad. Sci. USA 78 (1981) A J . b2.POP.'N iX. Q. R a R B XB S B B F A X wt Sc2601 I h is31 l j I I 10.1 kb Sc2605 i 6.1 kb nin5 B - adOd' MEo3 Xgt-XC' Sc2732 I-4 2.1 kb Sc2677 , -_ 1.3 kb Xgt4-Sc2601 B To hF E F T Fl E E HhT H K P X I# p F his3 Xgt9-Sc2601 T t E at MOOi C r'h E F MTM F II ,10 Xgt4-his3A -300 -200 -100 (5 +10 IT T T. GB R DR pid 13O3a C3 +_ F I Xgt9-his3A _IlL9 2670 imm2l 7 G YRX21 0 I, ~~~112779- I I I 227778 2755 2771 imm2l 2757 H I-,i YRX21-his3A 2781 _1~~~~~~1z T__j 2772 ~~~~~~27652671 FIG. 1. (A) Wild-type A.EcoRIsites are indicated by vertical lines, phage genes are indicated above the horizontal line, and A attachment site is indicated by POP'. (B) Agt-AC'. Mutated EcoR[ sites (x), nin5 FIG. 2. his3 deletions. The his3 region is drawn in the inverted deletion (o), EcoRI B-fragment deletion, and EcoRI C-fragment inver- orientation with respect to Fig. 1 and to previous publications (8, 13). sion are indicated (14). (C) Agt4-Sc2601 contains the left arm from Thishas been done so that his3 transcription proceeds from leftto right Aplac5, a 10.1-kb EcoRI insertion of his3 DNA (-, direction of tran- (indicated by arrow). All drawings are to scale. (a) Sc2601, the 10.1-kb scription indicated; see Fig. 2) (13). (D) Agt9-Sc2601. (E) int-mediated EcoRI fragment containing the his3 gene(18). BamHI (B), EcoRI (R), deletion of Agt4-Sc2601 that has one end point at att (13). (F) Deletion Xho I (X), and Sal I (S) sites (vertical lines) and mapping positions of of Agt9.Sc2601 (A end point varies). (G) YRA21 contains the imm2l various subcloned fragments. (b) Expanded view of Sc2677. Additional substitution (i.) and a 1.4-kb EcoRI fragment containing trpl and sites include Taq I (T), Hha I (h), Hinfl (F), Hae HI (E), HindIM (H), arsl cloned at the Sst II site near nin5 (box marked TA). (H) Recom- Kpn I (K), and Pst I (P). Positions of fragments (F, E, T) used for nu- binant of phages F and G used for transformation of yeast. clease S1 mapping experiments andthe his3-A1 deletion are indicated. (c) Expanded view of 5'-flanking region of hia3. The coordinate scale is definedsuch that positive numbers indicate transcribed nucleotides, phage (14). The survivors were screened for the presence of-an negative numbers indicate nontranscribed nucleotides, and zero in- intact his3 structural gene by the lytic "plaque without a lawn" dicates the 5' terminus of the stable his3 mRNA (8). Also shown are assay, (16). Conditions were chosen so that the parental phage the positions of two Mbo II (M) sites, the start of translation (box con- scored negative. Ten presumptive deletion mutants from each taining AUG), the start of transcription (box containing RNA), the 36- original stock were picked and screened for sequences homol- bp direct repeat in the mRNA leader (DR), the 12-bp palindrome (IR), ogous to YIp5-Sc2732 (ref. 17; Fig. 2) by plaque-filter hybrid- the Goldberg-Hogness T-A-T-A-A-A-T-A box (GB) (19), the Pribnow box used for expression inE.
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