Identification of intragenic suppressor mutations in a hybrid Eo/Sc eRF1
We previously found that a hybrid Eo/Sc eRF1 was unable to support growth when provided as the only source of eRF1 in yeast cells (Salas-Marco et al., 2006
). Dual luciferase readthrough assays (Keeling et al., 2004
; Salas-Marco and Bedwell, 2005
) revealed that Eo/Sc eRF1 efficiently recognized UAA and UAG as stop codons, but not the UGA codon. These findings led us to hypothesize that intragenic suppressor mutations that restore UGA recognition should also restore the growth of cells expressing Eo/Sc eRF1, thus identifying functional determinants of UGA recognition.
A centromeric plasmid carrying the SUP45 gene encoding the Eo/Sc hybrid eRF1 under SUP45 promoter control was subjected to random mutagenesis and a pooled mutant library was transformed into a sup45Δ strain that also carried a plasmid encoding the wild type SUP45 under GAL1 promoter control (). Transformants were selected at 30°C on plates containing glucose to shut off expression of the wild type SUP45 gene. Three independent suppressors that restored growth carried the same Cys to Ser substitution at codon 124 (C124S; S. cerevisiae eRF1 numbering) in the highly conserved YxCxxxF motif.
Figure 1 Mutagenesis of Eo/Sc eRF1. (A) The Eo/Sc eRF1 plasmid used for random mutagenesis and the Sc eRF1 plasmid under GAL1 promoter control used to support growth of the sup45Δ strain while screening for suppressors of the hybrid Eo/Sc eRF1. (B) Growth (more ...)
Mutations at C124 of the YxCxxxF motif restore UGA recognition by Eo/Sc hybrid eRF1
The Eo/Sc C124S eRF1 mutant restored cell viability, but was associated with a slow growth and cold-sensitive phenotype ( and ). To determine the efficiency of stop codon recognition by Eo/Sc C124S eRF1, a dual luciferase-based readthrough assay was carried out (Grentzmann et al., 1998
; Howard et al., 2000
; Keeling et al., 2004
; Salas-Marco and Bedwell, 2004
; Salas-Marco et al., 2006
). This assay monitors the efficiency of stop codon recognition, since efficient release factor binding should preclude suppression of the stop codon by a near-cognate tRNA. As controls, stop codon recognition was also measured in strains expressing either wild type Sc eRF1 or the original Eo/Sc hybrid eRF1 (). Stop codon recognition was efficient (<0.4% readthrough) at all three stop codons in the strain expressing wild type Sc eRF1. Since the Eo/Sc eRF1 (expressed under SUP45
promoter control) was unable to support cell viability, growth of the strain expressing this construct was maintained by a plasmid expressing wild type Sc eRF1 under GAL1
promoter control. This strain was grown in SM galactose medium, and then shifted to SM glucose to shut off wild type eRF1 expression. After growth for 6 more generations, the residual level of Sc eRF1 was <10% of normal (Salas-Marco et al., 2006
), which allowed us to estimate the stop codon recognition of Eo/Sc eRF1. We found that Eo/Sc eRF1 exhibited low readthrough (<0.7%) at UAA and UAG stop codons, but >10% readthrough at a UGA codon. These results confirmed our previous finding that Eo/Sc eRF1 efficiently recognizes UAA and UAG codons, but is severely compromised in UGA recognition.
Summary of readthrough and growth data for eRF1 variants used in this study
We found that the strain expressing Eo/Sc C124S eRF1 exhibited <0.4% readthrough at UAA and UAG stop codons, indicating that efficient recognition was retained at these stop codons (). Remarkably, only ~0.15% readthrough was measured at the UGA stop codon, which was less than the readthrough observed with wild type Sc eRF1. These results indicate that the C124S mutation successfully restored efficient recognition of the UGA stop codon without significantly compromising recognition of the UAA or UAG stop codons.
The cysteine residue of the YxC
xxxF motif in domain 1 is universally conserved among known eukaryotic eRF1 proteins (including variant code ciliate species) (Kim et al., 2005
; Kisselev et al., 2003
). To determine whether other amino acid substitutions at this position also restore UGA recognition by Eo/Sc eRF1, we tested each of the other 18 amino acids at this position. We found that only C124N supported cell viability, and it resulted in a cold-sensitive phenotype like the C124S mutation (). Readthrough assays carried out with strains expressing Eo/Sc C124N eRF1 revealed very low readthrough (~0.1%) at the UGA stop codon, and 0.2 to 0.4% readthrough at the UAA and UAG codons (). These results indicate that the C124N mutation, like C124S, is able to efficiently restore UGA stop codon recognition without compromising UAA and UAG recognition.
Figure 2 Efficiency of stop codon recognition mediated by Eo/Sc eRF1 suppressor mutants. (A) Alignment of TASNIKS and YxCxxxF motifs from Saccharomyces eRF1 and Euplotes eRF1. Numbering from Saccharomyces eRF1 is used. (B) Readthrough of stop codons measured in (more ...)
While characterizing the C124N mutation, we found a fast growing colony that had spontaneously acquired a second mutation, A75S (S. cerevisiae eRF1 numbering; corresponds to T75 in Sc eRF1). Interestingly, this mutation is located near C124 in the three-dimensional structure of eRF1. Strains expressing Eo/Sc eRF1 proteins with a combination of either the A75S/C124N or A75S/C124S mutations grew better at 30°C and 35°C than a strain expressing an Eo/Sc eRF1 with either C124 mutation alone (). Furthermore, the Eo/Sc A75S eRF1 supported cell growth as the sole source of eRF1. Readthrough assays carried out on strains expressing Eo/Sc A75S eRF1 showed very low readthrough (0.2%) at UAA and UAG codons, but 0.9% readthrough at UGA (). Thus, the A75S mutation restored UGA recognition less efficiently than the C124S or C124N mutations. However, the combination of the A75S and C124N mutations resulted in less than 0.2% readthrough at the UGA codon, and 0.2% to 0.3% readthrough at UAA and UAG codons. Given that growth is better when A75S is combined with either C124N or C124S, it appears that these mutations together optimize an aspect of eRF1 function not reflected by the readthrough assay.
Since changes in the steady-state level of the Eo/Sc eRF1 suppressors might influence the overall termination efficiency, we next examined the relative abundance of each Eo/Sc eRF1 by western blot analysis. We found that the steady-state level of eRF1 was not reduced by any of the suppressor mutations. In fact, the steady-state level of the hybrid eRF1 suppressors was elevated 2 to 3-fold in the strains that had regained UGA recognition (). These results are consistent with our recent finding that a novel regulatory mechanism increases steady-state eRF1 protein levels when translation termination is compromised in Saccharomyces cerevisiae (Kallmeyer and Bedwell, unpublished results).
TASNIKS mutations also restore UGA recognition and function cooperatively with C124S in the Eo/Sc hybrid eRF1
Previous studies have implicated the NIKS motif (residues 58 to 61 of S. cerevisiae
eRF1) in stop codon recognition (Frolova et al., 2002
; Seit-Nebi et al., 2002
). This motif is highly conserved in eRF1 proteins from standard code organisms, and crosslinking studies suggest that it is in close proximity to stop codons in the A site (Chavatte et al., 2002
). Furthermore, this motif is frequently altered in variant code organisms, suggesting that divergence from the consensus TASNIKS sequence may contribute to changes in stop codon recognition (Inagaki and Doolittle, 2001
; Knight et al., 2000
). In Euplotes octocarinatus
eRF1a (used in this study), this motif has the sequence TAES
IKS, which differs from the consensus TASN
IKS by the presence of a glutamic acid (E) at residue 57 and serine (S) at residue 58 (). To determine the importance of these variations on stop codon recognition, we introduced single amino acid changes in the Eo/Sc hybrid eRF1 (E57S or S58N) or a double mutant (E57S/S58N) that restored the standard TASNIKS motif. Viability assays indicated that the single mutations did not allow the growth of cells expressing these mutants as the sole source of eRF1. In contrast, the Eo/Sc eRF1 containing both mutations (Eo/Sc E57S/S58N eRF1) restored viability, but growth was again cold sensitive ().
We next asked whether combining mutations in the TASNIKS and YxCxxxF motifs could further improve growth of the strain expressing Eo/Sc eRF1. We found that strains expressing Eo/Sc eRF1 proteins containing either single TASNIKS mutation, E57S or S58N, in conjunction with the YxCxxxF mutation C124S mutation were viable and had better growth than a strain expressing the Eo/Sc C124S eRF1 alone (). The triple mutant, Eo/Sc E57S/S58N/C124S eRF1, exhibited the best growth. These results indicated that changes in the TASNIKS motif and C124 are cooperative with respect to cell growth. We also examined the affects of combining the E57S/S58N mutations with A75S in Eo/Sc eRF1. Viability assays showed that this combination of mutations also improved growth as compared to either of the two TASNIKS mutations or the A75S mutation separately. The E57S/S58N/A75S mutant grew less well than the E57S/S58N/C124S mutant, but better than the A75S/C124S mutant.
Readthrough assays carried out with a strain expressing Eo/Sc E57S/S58N eRF1 showed 0.2% readthrough at UAA and UAG, but 0.7% readthrough at UGA (). These results indicated that the E57S/S58N mutations were less efficient in restoring UGA recognition than the C124S mutation alone. However, the E57S/S58N/C124S triple mutant exhibited less than 0.2% readthrough at all three stop codons. This level of readthrough was lower than the Eo/Sc hybrid eRF1 carrying the two TASNIKS mutations at all three stop codons, and less than the hybrid eRF1 carrying the C124S mutation at the UAA and UAG codons. Again, these results suggest that the C124S and E57S/S58N mutations act cooperatively to fine-tune overall eRF1 function. Consistent with this premise, western blot analysis of the steady-state levels of these eRF1 proteins revealed a 2.3-fold excess of Eo/Sc C124S eRF1 and a 4-fold excess of Eo/Sc E57S/S58N eRF1, while the Eo/Sc E57S/S58N/C124S eRF1 level was reduced to a level only 1.3-fold above normal (). Since increased eRF1 levels correlate with a defect in translation termination, these results suggest that overall eRF1 function was better when all three mutations were present.
TASNIKS and C124 mutations reduce stop codon recognition by S. cerevisiae eRF1
Our results indicate that both the TASNIKS motif and C124 are important for UGA recognition by Eo/Sc eRF1. To determine the role of the corresponding residues in an eRF1 from a standard code organism, we introduced the C124S mutation and altered the TASNIKS motif to the TAESIKS element found in Euplotes eRF1 by introducing single (S57E or N58S) or double mutations (S57E/N58S) into domain 1 of Sc eRF1. Viability assays showed that Sc eRF1s with the single mutations, C124S, S57E or N58S could support cell viability, but all reduced the growth rate relative to a strain expressing wild type Sc eRF1 (). The double TASNIKS mutant (S57E/N58S) resulted in a more severe growth defect. Readthrough assays carried out with a strain expressing Sc C124S eRF1 showed 0.35% and 0.2% readthrough at UAA and UAG respectively, similar to wild type Sc eRF1 (). Readthrough at the UGA codon was less than 0.2%, which was about two fold less than the readthrough observed with wild type Sc eRF1. These results indicate that the C124S mutation makes UGA recognition more efficient in a standard code eRF1, as it does in the variant code Eo/Sc eRF1.
Figure 3 Efficiency of stop codon recognition mediated by Sc eRF1 carrying C124S or TASNIKS mutations. (A) Readthrough of stop codons measured in strains expressing Sc eRF1 with C124S or viable TASNIKS mutations. (B) Western blot quantitation of eRF1 levels from (more ...)
Readthrough assays of Sc eRF1 proteins carrying the S57E, N58S and S57E/N58S mutations showed a low level of readthrough (0.2 to 0.35%) at the UAA and UAG stop codons. However, 0.8% to 1.0% readthrough was observed at the UGA stop codon in strains expressing these mutant proteins, a ≥2-fold increase in readthrough compared to wild type Sc eRF1 (). This suggests that these changes in the TASNIKS motif of Sc eRF1 diminished the efficiency of UGA recognition, consistent with the observation that the reciprocal mutations that improved the TASNIKS homology in Eo/Sc eRF1 increased the efficiency of UGA recognition. The protein levels of all of these Sc eRF1 mutants were essentially normal ().
When we combined the S57E or N58S TASNIKS mutations with C124S in Sc eRF1, we found that none of the strains expressing pairwise combinations or the triple mutant were viable (). To determine the effect of the mutations on stop codon recognition, we carried out readthrough assays on strains expressing these mutant Sc eRF1 proteins in yeast cells after wild type Sc eRF1 had been depleted by growth in the absence of wild type Sc eRF1 for six generations. A strain constitutively expressing wild type Sc eRF1 was used as a positive control, while a strain depleted of eRF1 was used as a negative control (). The wild type strain exhibited 0.5% readthrough at the UAA stop codon in this series of experiments, while 5.3% readthrough was observed in the eRF1-depleted strain. The strain expressing Sc S57E/C124S eRF1 exhibited a wild type level of readthrough (0.6%) at the UAA codon. In contrast, the strains expressing Sc N58S/C124S eRF1 and S57E/N58S/C124S eRF1 resulted in 5.1% and 4.0% readthrough, respectively, at the UAA stop codon (8 to 10-fold higher than wild type eRF1). This level was similar to the readthrough observed following eRF1 depletion.
At the UAG stop codon, readthrough in the strain expressing wild type eRF1 was 0.2%, while readthrough in the eRF1-depleted strain was 4.7%. Readthrough in the strain expressing Sc S57E/C124S eRF1 was 1.1% (5 fold higher than wild type eRF1). Readthrough in strains expressing Sc N58S/C124S eRF1 or Sc S57E/N58S/C124S eRF1 was 4.8% and 3.9% respectively, again near the level observed following eRF1 depletion. At the UGA stop codon, 0.8% readthrough was observed in the strain expressing wild type Sc eRF1, while 18.4% readthrough was measured following eRF1 depletion. Readthrough at the UGA codon in strains expressing Sc eRF1 carrying a single TASNIKS mutation and C124S (S57E/C124S or N58S/C124S) was normal (0.6%), while readthrough in a strain expressing Sc eRF1 with both TASNIKS mutations and C124S (S57E/N58S/C124S) was 2.6%, or 4.3-fold higher than the strain expressing wild type Sc eRF1.
These results indicated that Sc S57E/C124S eRF1 exhibits only a partial defect in UAG recognition. In contrast, the strain expressing Sc N58S/C124S eRF1 had a severe defect in both UAA and UAG recognition, but retained efficient UGA recognition. This demonstrated that these two mutations were sufficient to convert Sc eRF1 to a UGA-only pattern of recognition. The Sc S57E/N58S/C124S eRF1 had a severe defect in UAA and UAG recognition, and also exhibited a partial defect in UGA recognition. This indicated that the S57 and N58 residues in the TASNIKS motif are important for UAA and UAG recognition, but are less important for UGA recognition. In contrast, the C124S mutation exerted a negative effect on UAA and UAG stop codon recognition when combined with the N58S TASNIKS mutation, indicating that C124 influences stop codon recognition in conjunction with this TASNIKS residue. Only when all three mutations are combined did UGA recognition deteriorate. When taken together, these results demonstrate that the TASNIKS and YxCxxxF motifs function cooperatively to mediate efficient stop codon recognition in Sc eRF1.
In vitro peptide release activity of Eo/Sc eRF1 suppressor mutants at UAA, UAG and UGA stop codons
We next examined the relative efficiency of the Eo/Sc eRF1 suppressors in mediating polypeptide release at different stop codons using a recently described in vitro
peptide release assay (Alkalaeva et al., 2006
). In this assay, mammalian pre-termination complexes (pre-TCs) containing peptidyl-tRNA in the P site and a stop codon in the A site were assembled on Met-Val-His-Cys (MVHC-STOP) mRNAs (encoding a MVHC tetrapeptide followed by either a UAA, UAG or UGA stop codon) using 40S and 60S ribosomal subunits, purified initiation and elongation factors, and aminoacylated tRNAs. Pre-TCs were purified by sucrose gradient centrifugation, and peptide release was initiated by the addition of eRFs. Since previous studies demonstrated that GTP hydrolysis by eRF3 is a prerequisite for efficient stop codon recognition and peptide release (Alkalaeva et al., 2006
; Salas-Marco and Bedwell, 2004
), peptide release induced by Eo/Sc eRF1 suppressors was investigated in the absence and in the presence of eRF3. Release of M-V-H-[35
S]C tetrapeptide as a function of time was monitored by scintillation counting of supernatants after TCA precipitation of reaction mixtures.
We first verified the ability of yeast eRFs (Sc eRF1 and Sc eRF3) to promote peptide release on mammalian pre-TCs. Incubation of pre-TCs for 20 minutes with saturating amounts of Sc eRF1 alone or in combination with Sc eRF3 in the presence of GTP resulted in nearly complete peptide release, just as with human (Hs) eRFs (). Release mediated by the combination of Sc eRF1 and Sc eRF3 was inhibited by the non-hydrolyzable GTP analog GMPPNP, again as with Hs eRFs (Alkalaeva et al., 2006
). To investigate the kinetics of peptide release, the concentrations of Sc eRFs were reduced, but release factors continued to be in excess over pre-TCs so that their recycling was not required. Like Hs eRF1, Sc eRF1 alone promoted slow peptide release, which was strongly increased by Sc eRF3 in the presence of GTP (, closed and open circles). These results indicated that the behavior of yeast and human eRFs was identical in this in vitro
system, which justified the use of mammalian pre-TCs to study the activities of yeast release factors.
Figure 4 Eo/Sc eRF1 mutants restore peptide release at the UGA stop codon. A) Endpoint peptide release assay with excess Sc eRF1 and Sc eRF3. B) Kinetics of peptide release with Sc eRF1 (+/− Sc eRF3). C) Kinetics of peptide release with Eo/Sc eRF1 and (more ...)
We next compared the rate of peptide release induced by Sc eRF1, Eo/Sc eRF1, Eo/Sc E57S/S58N eRF1, and Eo/Sc C124S eRF1 in the presence or absence of Sc eRF3. As in the case of Sc eRF1, slow peptide release at UAA and UAG stop codons promoted by the original Eo/Sc eRF1 hybrid alone was strongly stimulated by Sc eRF3•GTP (, closed and open squares). However, peptide release promoted by Eo/Sc eRF1 and Sc eRF3•GTP was slower than peptide release promoted by Sc eRF1 and Sc eRF3•GTP, which was most likely due to the heterogeneity of the system: even though mammalian pre-TCs could tolerate yeast release factors well, the use of the Eo/Sc eRF1 hybrid introduced an additional challenge. Consistent with the in vivo data, peptide release induced by Eo/Sc eRF1 on the UGA codon was slow even in the presence of Sc eRF3•GTP (, closed and open squares). Again, reflecting the in vivo situation, the E57S/S58N and C124S mutations in Eo/Sc eRF1 restored its activity at the UGA stop codon: the rates of peptide release on the UGA codon by Eo/Sc E57S/S58N eRF1 and Eo/Sc C124S eRF1 in the absence and in the presence of Sc eRF3•GTP (, closed and open triangles and diamonds, respectively) were similar to the rates of peptide release by Eo/Sc eRF1 on UAA and UAG codons (, closed and open squares). These in vitro data confirmed the in vivo results described above that the E57S/S58N and C124S mutations restore the peptide release activity of Eo/Sc eRF1 at the UGA stop codon.
The C124S mutation did not change the activity of Eo/Sc eRF1 at UAA and UAG codons, and as in the case of the original Eo/Sc eRF1 hybrid, the low activity of Eo/Sc C124S eRF1 alone was also strongly enhanced by Sc eRF3•GTP (, closed and open diamonds). However, the rate of peptide release on UAA and UAG codons by Eo/Sc E57S/S58N eRF1 alone was as high as the rates of peptide release on these codons by either Eo/Sc eRF1 or Eo/Sc C124S eRF1 in the presence of Sc eRF3•GTP, and was not stimulated further by Sc eRF3•GTP (, open and closed triangles). This suggested that in addition to restoring peptide release activity on the UGA stop codon, the E57S/S58N mutations also enabled Eo/Sc eRF1 to promote efficient peptide release in vitro at UAA and UAG codons independently of eRF3.
The Eo/Sc E57S/S58N eRF1 suppresses an eRF1 mutation that reduces eRF3 binding in vivo
After finding that the E57S/S58N mutations in Eo/Sc eRF1 enabled it to promote efficient peptide release at UAA and UAG codons independently of eRF3 in vitro
, we next examined whether these TASNIKS mutations also altered the eRF3 requirement for efficient stop codon recognition by eRF1 in vivo
. To test this, we used a modified form of Eo/Sc E57S/S58N eRF1, E57S/S58N/CΔ19, which carried a deletion of the last 19 amino acids (referred to as CΔ19). This deletion was previously shown to greatly reduce the ability of eRF1 to bind eRF3, resulting in a slow growth phenotype and increased readthrough at all three stop codons (Eurwilaichitr et al., 1999
; Kallmeyer et al., 2006
We found that a strain expressing CΔ19 eRF1 grew more slowly than strains expressing either wild type Sc eRF1 or Eo/Sc E57S/S58N eRF1, while a strain expressing Eo/Sc E57S/S58N/CΔ19 eRF1 had an even stronger growth defect (). When we examined the efficiency of stop codon recognition, we found that the Sc CΔ19 eRF1 strain exhibited elevated readthrough at all three stop codons (1.4% readthrough at UAA, 1.1% readthrough at UAG, and 6.6% readthrough at UGA stop codons) (). This general increase in readthrough caused by the CΔ19 mutation is consistent with a previous report (Eurwilaichitr et al., 1999
). In contrast, the Eo/Sc E57S/S58N/CΔ19 eRF1 strain exhibited < 0.2% readthrough at UAA and UAG codons (5 to 7-fold lower than in the Sc CΔ19 eRF1 strain), but 6.9% readthrough at the UGA codon (similar to the Sc CΔ19 eRF1 strain). These results indicate that this mutant protein retains efficient stop codon recognition at UAA and UAG codons despite a reduced ability to associate with eRF3, suggesting that the TASNIKS mutations result in a reduced requirement for eRF3 during the termination process at these two codons in vivo
. In contrast, recognition of the UGA codon was not restored to a comparable extent. Western blot analysis showed that the steady-state level of Sc CΔ19 eRF1 protein was elevated 11-fold relative to wild type eRF1, consistent with a strong defect in translation termination (). Interestingly, the Eo/Sc E57S/S58N/CΔ19 eRF1 level was 18-fold higher than wild type eRF1. This suggested that this mutant protein retained a severe defect in some aspect of the termination process.
Figure 5 Effect of reducing eRF3 binding on Eo/Sc E57S/S58N eRF1 function in vivo. A) Readthrough in strains carrying Sc eRF1, Sc CΔ19 eRF1, Eo/Sc E57S/S58N eRF1, or Eo/Sc E57S/S58N/CΔ19 eRF1. B) Western blot quantitation of eRF1 levels in strains (more ...)
Although our results demonstrated that both the TASNIKS and YxCxxxF motifs play cooperative roles in stop codon recognition, they are spatially separated in the surface of eRF1 domain 1, making it unlikely that both are in direct contact with the stop codon during the recognition process. Taking this into consideration, the finding that the TASNIKS mutations alter the eRF3 requirement for efficient stop codon recognition by eRF1 in vivo and in vitro suggests a potential alternative important role for the TASNIKS motif that is distinct from direct interaction with stop codons.