5' untranslated sequences are required for the translational control of a yeast regulatory gene.

104

116

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCNI, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCDI and mutations in four additional GCD genes which we designate GCDIO, GCDII, GCD12, and GCDI3. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::1acZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

Section: Resultsmentioning

confidence: 99%

“…Only one of these genes, GCDJ, was described previously (38). Each complementation group is represented by at least two independently (Fig.…”

Section: Discussionmentioning

confidence: 99%

Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

Harashima¹,

Hinnebusch²

1986

104

116

“…By standard methods, the deletion DNAs were introduced in a single copy at the his3 locus of strain KY466 (ura3-52 his3-A200 gcdl-1). The gcdl-J allele in this strain abolishes the translational control of gcn4 RNA and results in a constitutively high level of GCN4 protein (5,21). This situation causes induced levels of all coregulated genes even during growth in broth, and it is equivalent to conditions of amino acid starvation (22).…”

Section: Resultsmentioning

confidence: 99%

Two related regulatory sequences are required for maximal induction of Saccharomyces cerevisiae his3 transcription.

Struhl¹,

Hill²

1987

In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extending to -94 or beyond do not produce detectable levels of his3 mRNA. Small internal deletions that only remove the proximal regulatory sequence and a 1-base-pair deletion of the thymine residue at -99 abolish induction, but do not affect the basal level of transcription. These results indicate that the proximal copy between -99 and -94 is absolutely required for his3 induction, whereas the copy between -142 and -137 is required only for the maximal level of induction and is inactive by itself. From these and other observations, we suggest the possibility that these related regulatory sequences may be targets for two distinct proteins.

“…Sherman and Stewart (62) concluded from their study of cytochrome c mutants that yeast ribosomes cannot reinitiate, but in all of their mutants the upstream minicistron terminated very close to the ATG triplet that would have been used to reinitiate cytochrome c. That motif might be unfavorable for reinitiation in yeast, as it is in higher eucaryotes. Since upstream ATG codons, invariably followed by a terminator codon, have been found in a number of yeast mRNAs (18,24,42,50,56,66,67), it seems likely that yeast ribosomes either reinitiate or undergo leaky scanning, but neither mechanism has yet been established experimentally in yeast.…”

Section: Discussionmentioning

confidence: 99%

Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.

Kozak¹

1987

502

433

Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes reinitiate less efficiently as the intercistronic gap is widened.Eucaryotic ribosomes usually initiate at the AUG codon that lies closest to the 5' end of the mRNA (31). That tendency has been rationalized by postulating that the small ribosomal subunit binds initially at the capped 5' end of the mRNA and subsequently migrates to the AUG initiator codon, which is recognized more or less efficiently depending on nearby sequences. From a comparison of several hundred vertebrate mRNAs, GCCACCAUGG has been proposed as the consensus sequence for initiation in higher eucaryotes (30, 33, Kozak, submitted for publication). The contribution of every nucleotide in that motif has been confirmed by mutagenesis (36; Kozak, J. Mol. Biol., in press). The most highly conserved nucleotides are the purine, most often A, in position -3 (i.e., three nucleotides upstream from the AUG codon) and the G in position +4; the aforementioned mutational analyses confirmed that those two positions have the strongest influence on initiation. Thus, a potential initiator codon can usually be designated as "strong" or "weak" by considering only those positions.There are ways for eucaryotic ribosomes to initiate at internal sites in certain mRNAs, but those sites can be reached only by advancing from the 5' end; never, it seems, by direct internal binding. An early indication of the constraint against direct internal binding was the inability of eucaryotic ribosomes to bind to circular RNA templates (27,28), and a considerable body of other evidence has since been adduced (see Discussion). One mechanism by which ribosomes can reach an internal AUG codon is "leaky scanning," which occurs when the 5'-proximal AUG codon lies in an unfavorable context for initiation. Data consistent with leaky scanning have come from manipulating the sequences of synthetic constructs (35,36,40) and from analyzing naturally occurring viral mRNAs that are bifunctional (reviewed in references 38 and 39). A second mechanism for reaching an internal AUG codon operates when the upstre...