Molecular Analysis ofGCN3,a Translational Activator ofGCN4: Evidence forPosttranslationa l Control ofGCN3 Regulatory Function

GCN4encodes atranscriptional activator ofaminoacidbiosynthetic genesinSaccharomyces cerevisiae. The GCN3product isapositive regulator required forincreased synthesis ofGCN4protein inaminoacid-starved cells. GCN3appearstoactindirectly byantagonizing GCD-encoded negative regulators ofGCN4expression understarvation conditions; however, GCN3canalso suppresstheeffects ofgediandgcdl2 mutations under nonstarvation conditions. Theseresults implythattheGCN3product can promoteeither repression or activation ofGCN4expression depending on aminoacidavailability. We present a complete physical description oftheGCN3geneanditstranscript, plus measurements ofGCN3expression atthetranscriptional andtranslational levels underdifferent growth conditions. GCN3encodes a305-amino-acid polypeptide with no significant homology toanyother knownprotein sequence.GCN3mRNA contains noleader AUG codons, and no potential GCN4binding sites werefoundinGCN35'noncoding DNA.Inaccord withtheabsence ofthese regulatory sequencesfound atother genesinthegeneral control system, GCN3mRNA andaGCN3-lacZ fusion enzyme arepresent atsimilar levels underbothstarvation andnonstarvation conditions. Thesedatasuggest that modulation ofGCN3regulatory function inresponsetoaminoacidavailability occursposttranslationaliy. A gcn3deletion leads tounconditional lethality ina gcdl-1O1 mutant,supporting theideathatGCN3is expressed undernormal growth conditions andcooperates withtheGCD1product underthese circumstances tocarryoutan essential cellular function. We describe a point mutation thataddsthree aminoacids tothe carboxyl terminus ofGCN3,whichinactivates itspositive regulatory function required understarvation conditions without impairing itsability topromotefunctions carried outbyGCD12undernonstarvation conditions. Expression ofamino-acid-biosynthetic genesintheyeast Saccharomyces cerevisiae isregulated byatleast twomechanisms. Thefirst involves pathway-specific repression bythe aminoacidendproducts ofcertain pathways. A second mechanism, knownasgeneral aminoacidcontrol, leads to increased transcription ofatleast 30genesencoding enzymesinninedifferent pathways inresponse tostarvation foranysingle aminoacid. Theproducts ofnineGCN genes arerequired forderepression ofstructural genessubject to general control under starvation conditions. Theproducts of 12GCD genesarerequired forrepression ofthese genes undernormal growth conditions. Studies ofepistasis relationships amongregulatory mutations suggest that theproductsofGCNI,GCN2,andGCN3actindirectly aspositive effectors bynegative regulation ofGCDgeneproducts (for a review, seereference 19). GCN4,identified bythis genetic analysis asthemostdirect positive regulator inthegeneral control system, wasshowntofunction asatranscriptional activator bybinding toregulatory sequences located upstream fromstructural genessubject tothegeneral control