QUARTERLY Suppressors of translation initiation defect in hem12 locus of

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.

GTP and binding of the 5¢ end of mRNA to ribosomes is stimulated by eIF4F, eIF4A and eIF4B.The 43S preinitiation complex then scans the leader region of mRNA for the first downstream AUG codon that is a start site for translation in the majority of eukaryotic mRNAs.Once the AUG codon is found, eIF5 stimulates the hydrolysis of GTP bound to eIF2, the initiation factors are released and the 60S subunit joins the 40S subunit to form the 80S initiation complex and elongation of the peptide chain begins [1].
Eukaryotic ribosomes normally only select AUG codons as the start site for translation whereas prokaryotic translation can start by using alternative codons, such as GUG and UUG [2].In yeast, each possible mutation of AUG abolishes initiation of translation of HIS4 mRNA [3].Suppressor mutations in three genes called SUI1, SUI2, SUI3 were isolated that restore the His + phenotype of the his4 mutant despite the absence of the AUG initiator codon [4].The SUI1 gene product encodes a translation factor corresponding to the mammalian homolog, eIF1 [5,6].It copurifies with eIF3 and plays a role in translational accuracy [7,8].SUI2 and SUI3 encode the a and b subunits of the eIF2 complex, respectively [9,10].Mutations in the structural gene for eIF2g also influence the selection of the start site for protein synthesis [11].Thus, eIF1 and eIF2 control the recognition of the start codon by the ribosome and they influence the functioning of Met-tRNA i that directs the scanning ribosome to the start site [12].In many eukaryotic genes the first AUG in the mRNA sequence is not the translational start site of the main open reading frame (ORF).According to the most recent estimates, there are a few hundred genes in S. cerevisiae that have one or more small upstream ORFs (uORFs) that precede the main ORF [13].The uORFs usually inhibit, but sometimes stimulate, downstream translation [2,13].A major paradigm of eucaryotic translation regulation via uORFs is the GCN4 system of S. cerevisiae [14].eIF2 plays an important role in this regulation.
The HEM12 gene encodes uroporphyrinogen decarboxylase (Hem12p) [15], the fifth enzyme of the heme biosynthesis pathway [16].Molecular analysis of our collection of hem12 mutants revealed that the hem12-14 allele contains a mutation in the translation start codon ATG®ATA.This causes a lack of detectable Hem12p whereas a normal amount of hem12 mRNA is present [17].This defect results in the lack of growth of cells on media containing a nonfermentable carbon source, such as glycerol (gly -phenotype) and the accumulation of large amounts of porphyrins in the cell.Porphyrins are easily observed because of the red fluorescence (rf + phenotype) of cells under UV light [18].
In this report, we describe the isolation and characterization of extragenic and intragenic suppressors that can partially suppress the gly -phenotype of hem12-14 mutant.Mutational analysis of the HEM12 gene was also initiated to determine if uORFs present in the HEM12 leader sequence have a regulatory effect on HEM12 expression.
Yeast transformation was performed by the lithium acetate method [20].The transformants were recovered on glucose selective medium and the phenotype of the strains tested on YPGly medium [21].For biochemical analysis, cells were grown at 28 o C in YPG medium, supplemented with heme (15 mg/liter) or Tween 80 (0.2%) and ergosterol (30 mg/liter) for heme deficient mutants.For b-galactosidase assays, cells were grown in a selective medium containing 2% ethanol and 0.5% glucose.The X-gal indicator medium was prepared as previously described [22].
To isolate revertants, strains TZ21A or TZ21A/21C were plated on YPGly medium at approximately 10 7 cells/plate.Spontaneous revertants that fluoresce under UV at 366 nm were isolated after 3-4 days of incubation at 28 o C.

DNA preparation and manipulations
Escherichia coli strain DH5a and standard protocols were employed for DNA preparation, cloning and propagation [25].Yeast plasmid DNA for the transformation of E. coli was isolated as described by Rose et al. [21].All PCR amplifications were carried out with TaqI polymerase (Promega).DNA probes were radiolabeled by the random priming method with [a-32 P]dCTP (Amersham) using a kit from Boehringer.DNA sequencing was performed using an automatic ALF sequencer (Pharmacia).

Construction of yeast genomic library
Genomic DNA was isolated from RFR11-1A [21] and partially digested with endonuclease Sau3A to yield a maximum of fragments in the 6-10 kb range.The purified fragments were cloned into the BamHI site of the shuttle vector YCp50 [23].The resulting plasmid pools were used to transform E. coli by electroporation.After propagation on plates, plasmid DNA was extracted by alkaline lysis.Genomic DNA from other RFR mutants was digested with SphI and HindIII and fractionated by agarose gel electrophoresis.DNA fragments of approximately 2-3 kb in length were isolated and inserted into pUC18.Plasmids containing the HEM12 inserts were detected by in situ hybridization to a digoxigenin-la-beled HEM12 probe using DIG DNA Labeling and Detection Kit (Boehringer).

Mutagenesis of HEM12 leader sequence
The ATG initiation codons of the HEM12 uORFs were mutated by PCR mutagenesis.The SacI-EcoRV HEM12 fragment, encompassing 898 bp of the promoter and the first 243 bp of the coding region [15], was cloned into pBluescript KS and served as a template for reverse PCR amplification.In uORF1, the CAATGT (-279 to -274) sequence was replaced by AGATCT, introducing a BglII restriction site.In uORF2 the ATGAGG (-212 to -207) sequence was replaced by CCCGGG, introducing a SmaI site.In uORF3 the GAAATG (-184 to -179) sequence was replaced by GAATTC, introducing an EcoRI site.In uORF4 the GTGATG (-146 to -141) sequence was replaced by CTGCAG, introducing a PstI site.Two specific primers were used in the first PCR reaction, in which ATG codons of uORF2,3,4 were changed.The PCR product was cut with EcoRI, ligated and transformed into E. coli.Isolated plasmids, verified by restriction analysis, were used as a template in the second reverse PCR reaction to mutate uORF1.The PCR product was cut with BglII and ligated, giving pBShem12-1/4.The SphI-EcoRV 1116-bp fragment from this plasmid was inserted into the integrative lacZ fusion vector YIp358R [24] between SphI and SmaI sites.The resulting plasmid, YIphem12-1/4, was integrated into the URA3 locus of the S150-2B strain.Transformants were tested for b-galactosidase activity on X-gal indicator medium and in cell-free extracts as described [21].

RNA isolation and analysis
Total RNA (20 mg) was isolated as described [19] and fractionated by electrophoresis, transferred to nylon membranes and hybridized with radiolabelled probes by standard protocols [25].The HEM12 probe was the 1.27 kb PCR product [17].The same blot was hybridized with the XhoI-HindIII fragment of the ACT1 gene encoding actin for internal control of the amount of RNA loaded onto the gels.The autoradiograms were quantified by densitometry with an LKB UltroScan XL.

Low temperature spectra of whole cells and determination of porphyrin concentrations
Low temperature spectra of whole cells and porphyrins accumulated in the cells and excreted into the growth medium were determined as described previously [26].

Immunodetection of Hem12p
Total proteins (40 mg) extracted from yeast cells [27] were resolved by SDS-PAGE and transferred to nitrocellulose membranes.An alkaline phosphatase-coupled secondary antibody (Promega) was used to detect the anti-Hem12p antibody [28].

Isolation of external suppressors of hem12-14
Spontaneous reversion analysis was performed with the hem12 yeast strain with the ATA initiator codon.Gly + colonies that fluoresce under UV light (rf + ) were identified.This phenotype reflects partial Hem12p deficiency.The true revertants, ATA to ATG, have gly + rf -phenotype, as does the wild type strain.The frequency of spontaneous reversion to gly + rf + was 5 ´10 -6 , compared to the 1 ´10 -9 frequency of spontaneous reversion to the wild type.Twenty gly + rf + revertants were isolated, they are all recessive (Fig. 1) and constitute one complementation group, rfr1 (for red fluorescent revertant).The hem12-14 rfr1 suppressor strains accumulate less porphyrins than the hem12-14 mutant, as determined by low temperature spectra of whole cells and by porphyrin extraction analysis (not shown).A detailed genetic analysis of one revertant hem12-14 rfr1-1 confirmed that rfr1 is a single second site suppressor mutation and this strain was used in further studies.

Isolation of dominant suppressors of hem12-14
In search for new components of the translation initiation complex, the dominant suppressors of hem12-14 were isolated in the homoallelic hem12-14/hem12-14 diploid strain obtained by the cross of TZ21A and TZ21C.Three diploids, RFR8, RFR9 and RFR11 of phenotype gly + rf + were analyzed in detail.They all accumulate less porphyrins when compared to parental strain by low temperature spectra of whole cells (not shown).Analysis of progeny of RFR diploids revealed a tight linkage of suppressor mutations to hem12-14 (43, 32 and 60 tetrads analyzed, respectively).The haploid strain RFR11-1A was crossed to strains 117-8AR4, 117-8AR20 and 117-1AR7 that bear sui mutations.Analysis of spore clones from these crosses confirmed that RFR11 and sui are not allelic and showed that RFR11 and sui1-1 or SUI3-3 have additive effects.More Hem12p is observed by Western blot analysis in double mutant RFR11sui1-1 than in the respective single mutants (Fig. 3, lanes 5, 10-12).Consequently, RFR11sui1-1 and RFR11SUI3-3 do not fluoresce (rf -).RFR11 suppresses neither his4-303 nor his4-lacZ fusion, therefore is specific to hem12-14.RFR11 does not affect the steady-state level of HEM12 mRNA (Fig. 2, lanes 6-8).

Molecular analysis of suppressors linked to hem12
To characterize the RFR11 mutation, a genomic bank was prepared from DNA obtained from the RFR11-1A strain and plasmids complementing the gly -phenotype of hem12-14 mutant were isolated.All of four independent plasmids pKTE1, 3, 8, 9 contain the hem12 gene and only the subcloned fragments that contain the full hem12 gene complement the hem12-14 mutation.Sequencing of plasmid pKTE8 from -1016 to +266 nucleotides (+1 is A of ATG starting codon) of the hem12 gene identified an ATT ® ATG mutation at nucleotide -78 (Fig. 4).This mutation generates a new translational starting codon in frame with the downstream HEM12 ORF that extends the Hem12p N-terminus by 26 amino acids and is responsible for suppression.
sequenced.These mutations generate upstream additional ATG codons at positions -26 and -96, respectively, that are in frame with the downstream HEM12 ORF and allow the synthesis of longer forms of Hem12p (Fig. 4).

Translation of HEM12 ORF is not affected by uORFs
Examination of the HEM12 sequences reveals the presence of four uORFs in the 5¢ leader region.The longest transcript of the HEM12 gene contains a 22aa uORF1, an overlapping short 5aa uORF2, 11aa uORF3 and 7aa uORF4, that are positioned upstream with respect to the main ORF.The uORF1 and uORF2 are in frame with the main ORF.To determine whether uORFs regulate HEM12 expression, the ATG initiation codons -277, -212, -181 and -143 bp were mutated by PCR and hem12-1/4 allele generated.A plasmid carrying the hem12-1/4-lacZ translational fusion was integrated into yeast wild type strain.A strain containing the HEM12-lacZ integrated fusion was used as a positive control for lacZ expression.Independent transformants were tested for lacZ expression on X-gal indicator medium.Since the colony phenotypes of transformants were the same, the pooled cell-free extracts of twelve transformants were assayed in vitro for 186 M. Góra and others 2000  b-galactosidase activity.Mutations introduced in the HEM12 leader do not influence the b-galactosidase activity since hem12-1/ 4-lacZ mutants yielded activities of 9-14U, which were equivalent to that observed for the control (11U).

DISCUSSION
We have developed a genetic system designed to identify factors, acting either in cis or in trans, that suppress the effect of the mutant ATA initiation codon of the hem12 gene.The trans-acting rfr1 suppressors isolated are mapped to the SUI1 locus, encoding initiation factor eIF1.Mutant SUI3-3, encoding eIF2b, is also a suppressor of hem12-14.In contrast, sui2-1, another known suppressor of translation initiation defects, is unable to suppress the hem12-14 allele.SUI2 encodes eIF2a.All cis-acting suppressors analyzed partially restore the translation of hem12-14 by the generation of new, in frame AUG codons upstream of the hem12-14 mutation.We also determined that uORFs present in the HEM12 leader do not influence the efficiency of translation of the main HEM12 ORF.
Mutations in the SUI1 gene were first shown to affect start site selection, allowing translation to initiate at the non-AUG codon [5], but also to increase programmed -1 ribosomal frameshifting [8] and recently were shown to affect nonsense-mediated mRNA decay [29].Sui1p is suggested to contain an RNA-binding domain [30] and may function as a general regulator for RNA recognition in the processes of translation and mRNA decay.The mechanism of suppression by sui is common and results in an altered initiation start site [3,5,12], the UUG codon located at amino acid position three in the HIS4 coding region.Similar mechanism of suppression could be predicted for rfr1 and hem12-14; probably translation starts at the downstream UUG codon at amino acid position 11.
Why does sui2-1 not suppress hem12-14?The simplest explanation is that sui2 is unable to suppress hem12-14 because it is the weakest sui suppressor (8% of wt) [4].The other possibility may result from the special regulatory role of Sui2p.sui2-1 contains a mutation at the N-terminus of the a subunit of eIF2 [9].Ser-51 of eIF2a is phosphorylated by protein kinase Gcn2 and this phosphorylation mediates gene-specific translational control of GCN4 [31].Hyperphosphorylation of eIF2a at Ser-51 leads to down-regulation of global protein synthesis.Carboxyl-terminal serines of eIF2a are phosphorylated by casein kinase II and this modification is required for optimal function of eIF2 [32].It is possible that the hem12 mutant contains an abnormal phosphorylation status of the mutated form of eIF2a that does not allow suppression.
The hem12-14 mutation can be suppressed by intragenic mutations generating new, upstream AUG codons in frame with the HEM12 ORF.The suppression is partial, probably because of the suboptimal context around suppressor AUGs.The effect of suppressor AUGs is enhanced by the rfr1/sui1 suppressor and these two suppressors may operate independently or rfr1/sui1 further increases the initiation of translation from a new AUG.
Generally, translation initiates at the most 5¢-proximal AUG codon but translation initiation at the downstream AUG codon is possible by bypassing (leaky scanning) or reinitiation.These two processes depend on the position of the upstream AUG codons, the context of the two AUG codons [33] and the context of the uORF downstream sequences, respectively [13].Translation initiation from the upstream AUG codon with an optimal context can have a dramatic influence on translation initiation from the +1 AUG codon.The HEM12 initiator region 5¢-ACGCUAUGGGU-3¢ corresponds rather weakly to the yeast consensus start region, 5¢-AA/YAA/UAAUGUCU-3¢ [34].At least the start region of uORF2, 5¢-AAAAAAUGAGG-3¢ fits better to the yeast consensus and should efficiently initiate translation.However, we did not find an inhibitory effect of uORFs on translation of the main HEM12 ORF.As HEM12 mRNA has multiple 5¢ends, spanning positions -297 through -270 and-148 through -94 [19], the presence of uORFs can be limited to a subclass of mRNAs with longer 5¢ leader regions.The longer transcripts account for about 5% of total expression in the wt strain.Therefore, the effect of upstream ORFs on translation starting from the normal initiatior AUG codon could be limited and not physiologically significant.We cannot exclude its importance under some growth conditions.
In summary, translational suppressors of hem12-14 could function by at least two distinct mechanisms.These include initiating at codons other than AUG and generating a new AUG initiation codon upstream of the initial mutation.
We are grateful to T.F.Donahue for strains.

Figure 2 .
Figure 2. The rfr1 and RFR11 suppressor mutations do not affect the steady-state level of HEM12 mRNA.

Figure 4 .
Figure 4. Intragenic suppressors of hem12-14 generate new translational start codons.DNA sequences of the 5¢-region of the hem12 alleles.The ATG start codons are in bold.The mutant initiator codon of the hem12-14 allele is underlined.An arrow marks the position of the major initiation site of hem12 transcription.The first base of the normal ATG start codon is designated +1.