cis- andtrans-Acting Regulatory Elements oftheYeast URA3Promoter

Expression oftheyeast pyrimidine biosynthetic gene,URA3,isinduced three- tofivefold inresponsetouracil starvation, andthisregulation ismediated bythetranscriptional activator PPR1(pyrimidine pathway regulator 1).Inthisstudy, we haveanalyzed theregulatory elements oftheURA3promoter byDNaseI footprinting, using partially purified yeast cell extracts, bydeletion mutagenesis, andby5'-endmapping of RNA transcripts. TwoDNA-binding activities havebeendetected, andatleast fourdistinct cis-acting regions havebeenidentified. A region richinpoly(dA-dT) servesasan upstream promoter element necessaryforthe basallevel ofURA3expression. A 16-base-pair sequencewithdyadsymmetry actsas a uracil-controlled upstreamactivating site(UASURA) andshowsa specific binding onlywithcellextracts fromstrains overproducing PPR1.Thisinvitro binding doesnotrequire dihydroorotic acid, thephysiological inducer of URA3.TheTATA region appearstobecomposed oftwofunctionally distinct (constitutive andregulatory) elements. TwoG+A-rich regions surrounding this TATA boxbindan unidentified factor called GA-binding factor. The5'copy,GA,, isinvolved inPPR1induction andoverlaps theconstitutive TATA region. The3' region, GA2,isnecessaryformaximal expression. Neither ofthese GA sequencesacts asaUASinaCYCI-lacZ context. Thepromoters oftheunlinked butcoordinately regulated URAlandURA4genescontain highly conserved copies oftheUASURAsequence,whichprompted ustoinvestigate theeffects ofmany point mutations within this UASURAsequenceonPPR1-dependent binding. Inthis way,we haveidentified themostimportant residues ofthis binding site andfoundthat anonsymmetrical change ofthese bases issufficient toprevent the specific binding andtosuppresstheUASURAactivity invivo.Inaddition, we showedthatUASURA contains a constitutive activating element which canstimulate transcription fromaheterologous promoter independently ofdihydroorotic acidandPPR1. Yeastpromoters arecomposed ofmultiple elements that arenecessaryfortheregulation, frequency, andaccuracyof transcriptional initiation (reviewed inreference 44).Although asimple promoter could contain jUst oneofeachkind ofelement, mostSaccharomyces cerevisiae promoters are more complex. Upstreamelements resemble mammalian enhancer sequencesinthattheyfunction ineither orientationatvariable distances withrespect tootherpromoter elements andmRNA initiation site(s) butnevertheless are incapable ofactivating transcription whenplaced downstreamofthetranscription startsite(s) (13,15). TheTATA elements arenecessarybutnotsufficient fortranscriptional initiation ofmostgenes.Thedistance ofthese sequences fromthestartsite differs fromgene togene,andmultiple TATA sequencesareoften foundinthe5'noncoding region ofyeastgenes.Two typesofTATA sequenceshavebeen identified forHIS3.Oneofthem(Tc)behaves like aconstitutive element, andtheother one (TR)isnecessaryfor transcriptional induction underconditions ofaminoacid starvation (43). Theinitiator element (I)isusually located near themRNA startsitebutdoesnotplaya rolein determining themRNA level. Thespacing betweenthe TATA boxandIseems tobeimportant butcan varyfrom40 to120basepairs (32). Twotypesofelements involved inthe regulation ofthebasalleveloftranscription havebeen characterized: (i) a region richinpoly(dA-dT) inthecaseof theHIS3andPET56genes(42) and(ii) acis-acting region of theHIS4promoter, necessaryforthebinding oftwo pro