A Dispensable Yeast Ribosomal Protein Optimizes Peptidyltransferase Activity and Affects Translocation*

John DresiosDagger, Panagiotis Panopoulos, Katsuyuki Suzuki§, and Dennis Synetos

From the Laboratory of Biochemistry, School of Medicine, University of Patras, 26110 Patras, Greece and § Department of Chemistry & Environmental Technology, Kinki University, Hiroshima 739-2116, Japan

Received for publication, July 26, 2002, and in revised form, October 21, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (kcat/Ks) corresponding to the rate of peptide bond formation; this kcat/Ks was lowered 3-fold to 1.15 min-1 mM-1 in the L41 mutant compared with 3.46 min-1 mM-1 in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 µM EF2 compared with 0.02 µM for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 µM of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 µM in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The ribosome is a large macromolecular machine that consists of a large number of proteins and several molecules of ribosomal RNA (rRNA). The ribosome is responsible for the translation of the genetic message, which results in protein synthesis.

The crystal structures of the 30 S (1, 2) and 50 S (3, 4) ribosomal subunits, and the intact 70 S ribosome (5) are contributing to a better understanding of ribosomal function. It has now been confirmed that rRNA plays the major role in ribosomal structure and function, including the two most important activities of the ribosome: the decoding process and the peptidyltransferase activity. The 16 S rRNA of the small ribosomal subunit is responsible for the decoding process, the selection of the cognate tRNA (6). The central loop of domain V of 23 S rRNA of the large ribosomal subunit is responsible for the catalytic activity of the ribosome (7, 8). The search is still underway to identify a critical site or nucleotide(s) of 23 S rRNA involved in the catalysis of peptide bond formation (9-13). It is still possible that the rate of peptide bond formation is influenced by structural rearrangements of RNA in the peptidyltransferase center that would affect the positioning of the substrates (14).

Ribosomal proteins offer structural support to the ribosome by stabilizing and orienting the ribosomal RNA into a specific, active structure (15). Also, they are crucial for the assembly of functional ribosomes (16). Several ribosomal proteins of the small subunit (17-20) and at least one of the large subunit (21) appear to affect the decoding process. Moreover, some ribosomal proteins appear to influence the peptidyltransferase activity of the ribosome (22-24).

Yeast ribosomal protein L41 is the smallest and the most basic eukaryotic protein. Its open reading frame is composed of only 25 amino acids, 17 of which are arginines or lysines (25). L41 is highly conserved in eukaryotes and it is present in certain archaea, e.g. Methanococcus jannaschii, but not in eubacteria. A protein with similar properties is present in certain thermophilic eubacteria, e.g. Thermus thermophilus (26). In Saccharomyces cerevisiae, L41 is encoded by two genes, RPL41A and RPL41B (25). In a recent study, the properties and the translation of the mRNAs encoding L41 were investigated (27). It was found that the L41 mRNAs are translated exclusively on monosomes and the entire translation process from initiation through to termination occurs in about 2 s. However, as with most ribosomal proteins, the function of L41 remains unknown (27).

There is evidence that suggests that protein L41 may possess extraribosomal activity; it associates directly in vitro with subunit b of protein kinase CKII, although it is not a substrate for CKII phosphorylation. However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIa by CKII. Additionally, L41 enhances the autophosphorylation of CKIIa (28). These data indicate that L41 associates with CKII and can modulate its activity toward a specific substrate(s).

In the present work we tried to gain insight into the role of protein L41 in protein synthesis. For this purpose we examined two yeast strains. The first lacked the two genes encoding L41. The second strain lacked L41 in addition to the absence of L24 and L39, two eukaryote-specific proteins studied recently (21, 24). The fact that the cells remained viable upon removal of the two genes encoding L41 in addition to the absence of the two genes encoding L24 and the single gene encoding L39, allowed for the first time the study of a quintuple mutant in yeast as well as the study of the role of L41 in combination with the effects of the other two proteins. Our results suggest that a dispensable ribosomal protein such as L41 may affect in varying degrees several important functions of yeast ribosomes confirming the interconnectedness of these ribosomal activities.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Construction of Gene Disruption Plasmids-- A 1.1-kb EcoRI-digested DNA fragment that contains S. cerevisiae RPL41A (25) was subcloned into pUC118 vector and designated as pYL41A1 (Fig. 1). The open reading frame of RPL41A was completely deleted from pYL41A1 and a BglII restriction site was created by PCR according to Eberhardt and Hohmann (29). The primers used were 5'-GGAGATCTAAGCGGATTATGAGTAAATAAC-3' and 5'-GGAGATCTTCGATTGAATCGATGTGGTC-3'. An amplified DNA fragment was digested by BglII and selfligated to obtain pYL41A1dCOD-Bg. A 3.1-kb BglII fragment from Yep13 that contains the LEU2 gene, or a 3.8-kb BglII-BamHI fragment from pNKY51 bearing a hisG-URA3-hisG cassette (30) was inserted into the BglII site of pYL41A1dCOD-Bg to form plasmids pYL41A1dCOD-LEU2 or pYL41A1dCOD-URA3, respectively. A 2.9-kb EcoRI-digested DNA fragment that contains RPL41B (25) was subcloned into pUC118 vector and designated as pYL41B1 (Fig. 1). Plasmid pYL41B1dCOD-URA3 was constructed similarly as described above. The two primers used were 5'-GGAGATCTTGGAATTAAGTCGATATGAC-3' and 5'-TCCAAATAAGCGGATTGAGAG-3'. The latter does not have a BglII site. Instead, a BglII site located 134 bp downstream of the termination codon of RPL41B was used for inserting the hisG-URA3-hisG cassette.


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Fig. 1.   Construction of plasmids carrying intact and disrupted RPL41A (A) and RPL41B (B) genes. Probe A, a 1.0-kb EcoRI fragment of pYL41A1dCOD-Bg; probe B, a 2.0-kb EcoRI + BglII fragment of pYL41B1dCOD-Bg. Restriction sites: E, EcoRI; B, BglII.

Yeast Transformations and Gene Disruptions-- Yeast transformations were performed by a lithium acetate method (31). Plasmids pYL41A1dCOD-LEU2 or pYL41A1dCOD-URA3 were digested with ApaLI and SacI and used to transform yeast cells, resulting in Delta rpl41a strains. Plasmid pYL41B1dCOD-URA3 was digested with EcoRI and transformed into yeast cells to generate Delta rpl41b strains.

Construction of Yeast Strains-- Strains used are listed in Table I. Two genes for ribosomal protein L41 were successively disrupted in the homozygous diploid YPH501 (32) to obtain YKS99. Sporulation of YKS99 gave rise to four viable spores even from tetratype tetrads indicating that ribosomal protein L41 is not essential for cell viability. Haploid cells from one of the tetratype tetrads were isolated and designated YKS99-2A to YKS99-2D.

Strain 2A-J809 lacking the two genes for ribosomal protein L24 (33) was crossed with strain YAS282, in which RPL39 had been disrupted (34), and a progeny 38C was isolated. This strain was created and kindly provided by Dr. Irina L. Derkatch, Department of Biological Sciences, University of Illinois, Chicago, IL. To reuse the ura3 marker, a stable ura3 mutant of 38C was selected on 5-fluoroorotic acid media (YKS100). RPL41A in YKS100 was replaced with hisG-URA3-hisG cassette to create a new strain (YKS101). A ura3 mutant of YKS101 was selected on 5-fluoroorotic acid media again (YKS103). RPL41B in YKS103 was replaced with the hisG-URA3-hisG cassette again to create YKS121. In each step, Southern analysis was performed to confirm the gene disruption. Strains YKS100, YKS101, and YKS103 are omitted from Table I.

Biochemical Procedures-- Experiments to measure the sensitivity of cells to paromomycin, the preparation of a yeast cell-free system for translation in vitro, the translation of poly(U) templates in vitro, the P- and A-site binding studies as well as the time course measurements of polyphenylalanine synthesis, the sucrose gradient analysis, and the preparation of ribosomal proteins were all performed as described recently (21).

Two-dimensional Polyacrylamide Gel Electrophoresis-- Ribosomal protein L41 tends to be lost during two-dimensional gel electrophoresis (25). To obtain the spot corresponding to L41, the electrophoresis was carried out as follows: the first dimension was run in 4% polyacrylamide and 6 M urea at 3 mA/tube for 3 h from pH 4.0 to pH 5.0. The second dimension was run in 18% polyacrylamide and 6 M urea at 20 volts for 24 h. Gels were stained with Coomassie Brilliant Blue R-250 to visualize the proteins.

Formation, Isolation, and Extraction of Complex C-- Unwashed or, alternatively, high salt-washed ribosomes, soluble protein factors, and Ac1-[3H]Phe-tRNA from yeast, were prepared according to recently described methodology (24). The donor Ac-[3H]Phe-tRNA was 270 A260/ml and contained 14 pmol of [3H]Phe charged/1 A260 tRNA and 262,000 cpm incorporated/1 A260 tRNA. Complex C, i.e. the Ac-[3H]Phe-tRNA·poly(U)·80 S ribosome complex, was formed from unwashed or high salt-washed ribosomes in a binding mixture (0.2 ml) containing: 80 mM Tris-HCl, pH 7.4, 160 mM ammonium chloride, 11 mM magnesium acetate, 2 mM spermidine, 6 mM beta -mercaptoethanol (binding buffer), as well as 0.4 mM GTP, 30 A260/ml of 80 S ribosomes, 0.4 mg/ml poly(U), and 16 A260/ml Ac-[3H]Phe-tRNA from yeast. Following incubation for 16 min at 30 °C, the reaction was stopped by placing the binding mixture in ice. The solution was immediately filtered through a cellulose nitrate filter disk under vacuum with three 4-ml portions of binding buffer. The filter disks with the adsorbed complex C were gently shaken for 30 min at 5 °C in binding buffer containing 0.05% Zwittergent 3-12 (1.8 ml/whole disk). The extraction was stopped by removing the filters and the complex C containing Zwittergent 3-12 extract was used in the puromycin reaction.

Puromycin Reaction-- The reaction between the Ac-[3H]Phe-tRNA of complex C in the Zwittergent 3-12 extract and puromycin (puromycin reaction) was carried out as described recently (24). Zwittergent 3-12 extract (0.9 ml) was preincubated for 5 min at 30 °C. Then, puromycin (0.1 ml) at the desired concentrations was added and the puromycin reaction proceeded for the indicated time intervals and stopped with 1.0 ml of 1.0 N NaOH. If N0 represents the total radioactivity of bound donor (e.g. Ac-[3H]Phe-tRNA), the percentage (x) of the bound donor that was converted to product P (Ac-[3H]Phe-puromycin) was calculated by dividing P by N0 and multiplying by 100. Each percentage (x) was corrected, first, by dividing its value with factor A (A = C/C0, where C and C0 are the amounts of surviving complex C in binding buffer at the end of each incubation period and at zero time, respectively), and, second, with the extent factor alpha . The extent factor is determined when complex C is allowed to react completely, at any concentration of puromycin. By the first correction (x/A), the parallel inactivation of complex C during the puromycin reaction is subtracted, whereas by the second correction the intervention of any species other than complex C is erased as if 100% of the bound Ac-[3H]Phe-tRNA were reactive toward puromycin. Thus, the experimental values of x were corrected by factor Aalpha , i.e. x' = x/Aalpha .

Resistance to Cycloheximide-- The resistance of cells toward cycloheximide and the polyphenylalanine synthesis activity in the presence of increasing concentrations of cycloheximide were determined as described recently (24).

Translocation-- The reaction mixture for translocation (0.1 ml) was composed from the same binding buffer as that used for the formation of complex C, as well as 0.4 mM GTP, 30 A260/ml of ribosomes, 0.4 mg/ml poly(U), and tRNAPhe at a ratio of 4:1 to ribosomes. At this ratio, all P-sites were occupied by tRNA after 30 min at 30 °C. Then, 13 A260/ml yeast Ac-[3H]Phe-tRNA were added and Mg(CH3COO)2 was raised to 15 mM. Reincubation followed for 10 min at 30 °C to form a poly(U)-programmed ribosomal complex in which P-sites were filled with tRNA and A-sites with Ac-[3H]Phe-tRNA.

The reaction was stopped by placing the binding mixture in ice. Filtering, washings, and extraction of the ribosomal complex into binding buffer containing 0.05% Zwittergent 3-12 at 5 °C were carried out as described previously (24). Zwittergent 3-12 extract was preincubated for 2 min at 30 °C. Also, elongation factor 2 (EF2) prepared from rabbit reticulocytes (35) or soluble protein factors (SPF) prepared from yeast (24) were preincubated with 1 mM GTP, 40 mM phosphocreatine, and 40 µg/ml creatine phosphokinase for 15 min at 37 °C. The enzymatic translocation reaction was started when GTP-activated EF2 at 0.02 µM or SPF at 0.05 mg/ml were added to the pretranslocation Zwittergent 3-12 complex and allowed to react for the indicated time intervals. Single-turnover translocation (EF2: active ribosomes = 9:1) was monitored by reaction with 2 mM puromycin (3 × Ks) for 4 min (7 half-lives) at 30 °C. The puromycin solution contained cycloheximide at a final concentration of 8 µM to ensure that no translocation takes place during the puromycin reaction. Nonenzymatic translocation was measured in the absence of elongation factors.

For concentration dependence experiments, increasing concentrations of EF2 or SPF were added to the pretranslocation Zwittergent 3-12 complex and reacted for 1 min after which translocation was again monitored by reaction with puromycin. Finally, to determine the sensitivity of the various strains toward cycloheximide, the antibiotic was added at the appropriate concentrations together with the EF2 or SPF fractions, the reaction was allowed to proceed for 1 min and translocation was again monitored with the puromycin reaction.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ribosomal Protein L41 Is Dispensable in the Yeast-- By obtaining the YKS99-2C isolates (Table I), we established that yeast cells lacking the two genes that encode ribosomal protein L41 are viable. These cells grew in YPD (1% yeast extract, 2% bactopeptone, 2% D-glucose) with a doubling time of 1.8 h, i.e. 10% longer than the doubling time of 1.6 h required for the wild type (YKS99-2A). Of the strains carrying only one disrupted copy of RPL41, the Delta A strain had roughly the same doubling time as the control, whereas the Delta B strain had a doubling time of 1.8 h.

                              
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Table I
S. cerevisiae strains constructed for this study

The absence of RPL41A and -B from strain YKS99-2C was demonstrated by Southern blot analysis (Fig. 2) and the absence of L41 protein from the ribosomes of this strain by two-dimensional polyacrylamide gel electrophoresis (Fig. 3B).


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Fig. 2.   Southern blot analysis. Yeast genomic DNA cut with EcoRI was electrophoresed, transferred to nylon membranes, and hybridized to probe A and probe B simultaneously. Bands at 1.1 and 2.9 kb represent RPL41A and RPL41B, respectively, in wild-type strain YKS99-2A (lane 1). Two bands at 2.1 kb represent the replacement of RPL41A by rpl41a::LEU2 and a band at 6.5 kb represents the replacement of RPL41B by rpl41b::hisG-URA3-hisG, in strain YKS99-2C (lane 2). Bands at 2.2 and 6.5 kb represent the replacement of RPL41A by rpl41a::hisG and RPL41B by rpl41b::hisG-URA3-hisG, respectively, in the quintuple mutant strain YKS121 (lane 3).


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Fig. 3.   Two-dimensional gel electrophoresis of 80 S ribosomal proteins. A, YKS99-2A strain (wild type). B, YKS99-2C strain (-L41). C, YKS121 strain (-L41-L24-L39 or quintuple mutant). Details are mentioned under "Materials and Methods." In each case, the presence or absence of proteins L41, L24, and L39 is indicated by arrows.

Ribosomal protein L41 is the smallest and the most basic eukaryotic protein with a pI = 12.8. Consequently, it eluted close to the solvent face giving a not so clearly stained spot (Fig. 3A). A similar behavior was observed for its orthologous ribosomal protein Thx from T. thermophilus (26).

A Mutant Strain Carrying Deletions of Five 60 S Ribosomal Protein Genes Encoding Ribosomal Proteins L24, L39, and L41 Is Viable-- By obtaining the YKS121 isolates (Table I), we established that yeast cells simultaneously lacking all five genes encoding the L24, L39, and L41 ribosomal proteins are viable. However, this quintuple mutant strain was characterized by significantly reduced growth: its doubling time in YPD was 5.2 h, i.e. three times longer than the wild type. By comparison, the doubling time of the triple mutant strain 38C, in which the two genes for L24 and the single gene for L39 are missing, was 4.1 h.

Deletion of L39 caused cold sensitivity (34). In agreement with this observation the quintuple mutant strain YKS121, which lacks L39, is cold-sensitive, i.e. it did not grow when spotted on YPD at 20 °C. The concomitant absence of the five genes in strain YKS121 was determined by Southern blot analysis (Fig. 2) and the absence of the corresponding proteins L24, L39, and L41 by two-dimensional polyacrylamide gel electrophoresis (Fig. 3C).

Polyphenylalanine Synthesis Activity of Wild-type and Mutant Ribosomes-- Using in vitro poly(U)-dependent polyphenylalanine synthesis, we determined the activity of wild-type and mutant ribosomes (Table II). All ribosomes were dependent on the presence of soluble protein factors. In their absence, the ribosomes were unable to polymerize phenylalanine. As wild type, we routinely used ribosomes from strain YKS99-2A, because they exhibited the same polyphenylalanine synthesis activity as well as error frequency as those from strains 2D-J809 or YAS43, which are wild type to strains 2A-J809 and YAS282, respectively (Table I). Mutant ribosomes lacking L41 showed the same extent of polyphenylalanine synthesis activity as wild-type ribosomes (Table II). Moreover, ribosomes from the quintuple mutant YKS121, despite a 3-fold slower growth rate, displayed the same extent (Table II) but lower initial rates (not shown) of polyphenylalanine formation compared with the wild-type ribosomes, similar to the values reported for ribosomes from the triple mutant (21). Thus, the absence of L41 from the YKS99-2C or YKS121 strains does not affect polyphenylalanine synthesis.

                              
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Table II
Polyphenylalanine synthesis activity and error frequencies in vitro for wild-type and mutant ribosomes
End-point incorporation after a 32-min incubation.

Effect of Protein L41 on Translational Accuracy-- The cell-free system also allowed measurement of the misincorporation of the near cognate amino acid leucine with poly(U) as template. The accuracy of translation was determined by the error frequency (20). As shown in Table II, the error frequency of the wild-type strain YKS99-2A was 26 errors every 104 codons. This error rate is almost identical to that reported recently for 2D-J809, another wild-type strain (21) and it is within the range reported in the literature (36, 37).

The error frequency of the L41 mutant strain was slightly decreased compared with the wild type. The error frequency of the quintuple mutant was also slightly decreased compared with the triple mutant. The substantially increased error frequencies of the quintuple and triple mutants over that of the wild type (Table II) were previously shown to be caused by the absence of L39 (21).

This trend was confirmed in the presence of paromomycin, which is an error-inducing antibiotic. This antibiotic has been found to stimulate phenylalanine incorporation in wild-type and mutant ribosomes (20). Indeed, paromomycin at 50 µM increased the in vitro error frequency in all strains. However, the increases in the error frequency were lower for strains lacking L41 than for strains carrying L41 (Table II). We conclude that the absence of L41 causes slight hyperaccuracy.

Strains Lacking L41 Show Increased Resistance to Paromomycin-- Hyperaccuracy has been usually associated with increased resistance of cells to antibiotics such as paromomycin and vice versa (38). We tested whether the lower error frequencies shown by the L41 and quintuple mutants compared with the wild-type and triple mutants, respectively, were accompanied by increased resistance of the cells toward paromomycin. Cells were grown in YPD at 30 °C to an A660 of 0.9 that was taken as 100%. The growth of the wild-type strain was inhibited by 50% at 185 µM paromomycin. Deletion of both L41 genes resulted in increased resistance of the cells toward paromomycin (250 µM for 50% inhibition). Likewise, 50% inhibition of growth of the quintuple mutant strain in which L41 is absent was observed at 71 µM paromomycin compared with only 50 µM for the triple mutant in which L41 is present. The decreased resistance toward paromomycin of the triple mutant compared with the wild type is caused mainly by the absence of L39 (21).

P- and A-site Binding-- Under the conditions employed, Ac-Phe-tRNA was preferentially bound to the P-site. There was no change in the P-site binding of the Ac-Phe-tRNA between L41 mutants and wild type or between quintuple and triple mutants (Table III). The slight decrease in the P-site binding observed for either the quintuple or the triple mutants compared with wild type is because of the absence of L24 from these two strains (21).

                              
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Table III
Peptidyltransferase activity and binding capacities of P- and A-sites of wild-type and mutant ribosomes

Following full occupation of the P-site by deacylated tRNA, we determined the A-site binding by adding Phe-tRNA and measuring the radioactivity of cellulose nitrate filter disks. Table III shows the A-site binding capacities of the mutant strains expressed as percent of the radioactivity contained in the Phe-tRNA·80 S·poly(U) complex of wild-type strain YKS99-2A. Strain YKS99-2C, i.e. the strain lacking L41, showed an A-site binding slightly lower than the wild-type levels. Likewise, the quintuple mutant strain showed an A-site binding slightly lower than that of the triple mutant. With regards to the higher A-site binding shown by the quintuple and triple mutants over the wild type, it is caused by the absence of L39 (21) and this is in agreement with the finding that these two strains are also prone to translational errors (Table II).

Activity of the Wild-type and Mutant Ribosomes in Peptide Bond Formation-- Complex C, i.e. the Ac-[3H]Phe-tRNA· poly(U)·80 S ribosome complex, contained about 49% of the input Ac-Phe-tRNA (3.2 A260 units or 44.8 pmol of Ac-Phe-tRNA/0.2 ml of binding mixture), because only 49% of the input of radioactivity was adsorbed on the cellulose nitrate filter disk. Thus, complex C contained 22.0 pmol of Ac-Phe-tRNA. Assuming a 1:1 combination, this complex C also engages 20.5% of the ribosomes added (6.0 A260 units or 107.3 pmol). Hence, 0.205 molecules of Ac-Phe-tRNA are bound per molecule of poly(U)-programmed ribosomes.

The catalytic activity of eukaryotic peptidyltransferase was determined with the puromycin reaction.
(Eq. 1)
An advantage of this reaction is that it is carried out in two steps. In this way all reactions involved in the binding of the donor Ac-Phe-tRNA to the ribosomes and the formation of ternary complex C (step 1) have been separated from peptide bond formation (step 2). The reaction between the Zwittergent-extracted complex C (C) and excess puromycin (S) proceeded as a pseudo-first order reaction in which the product (P) is Ac-Phe-puromycin and C is converted to C', which cannot re-form complex C (Equation 1). Thus, each catalytic center of peptidyltransferase works only once. The rate constant (k3) gives the reactivity of the P-site-bound donor Ac-Phe-tRNA, and it is equal to the catalytic rate constant (kcat) of peptidyltransferase. Complex C from wild-type or mutant strains was remarkably stable because less than 5% of the bound Ac-Phe-tRNA was dissociated after 16 min. Nevertheless, the amount of donor that was converted to product was corrected by dividing its value with dissociation factor A as well as with extent factor alpha  (see "Materials and Methods").

Typical first-order time plots for Ac-[3H]Phe-puromycin formation were linear up to more than 90% depletion over a wide range of puromycin concentrations. The slopes of these straight lines give the kobs values at each puromycin concentration (not shown).

The apparent rate constant kobs is a function of [S] and it is given by Equation 2.
k<SUB><UP>obs</UP></SUB>=k<SUB><UP>cat</UP></SUB>[<UP>S</UP>]/(K<SUB>s</SUB>+[<UP>S</UP>]) (Eq. 2)
Equation 2 predicts that a plot of the reciprocal of the experimental kobs versus the reciprocal of the puromycin concentration (double reciprocal plot) should be linear. The values of kobs obtained from the wild-type strain were fitted in Equation 2 from which the double reciprocal plot gave kcat = 1.80 min-1 and Ks = 0.52 mM (Fig. 4 and Table III).


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Fig. 4.   Peptidyltransferase activity of wild-type and mutant ribosomes. Double reciprocal plots (1/kobs versus 1/[puromycin]) for Ac-[3H]Phe-puromycin formation, using complex C prepared with ribosomes from the (black-square) wild-type strain (black-down-triangle ) -L24-L39 or triple mutant strain (black-triangle) L41 mutant strain or () -L24-L39-L41 or quintuple mutant strain. From these plots, the Ks and kcat values for each strain are determined.

The reaction of complex C from all mutant strains with puromycin followed also pseudo-first order kinetics and the logarithmic time plots were linear (not shown). The double reciprocal plots were also linear (Fig. 4). From these plots it was calculated that the first-order catalytic rate constants (kcat) for the L41, triple, and quintuple mutants are 0.61, 0.83, and 0.51 min-1, respectively (Table III). The Ks remained the same. Ratio kcat/Ks is a second-order rate constant that is an accurate measure of the ribosomal peptidyltransferase activity of each strain. Thus, the L41 mutant formed peptide bonds at one-third the rate of the wild-type strain (kcat/Ks = 1.15 versus 3.46 min-1 mM-1). Also, the decrease in the kcat/Ks of the quintuple mutant compared with that of the triple mutant (kcat/Ks = 0.88 versus 1.48 min-1 mM-1) is further confirmation that the absence of L41 lowers the peptidyltransferase activity of the ribosome. Both kcat and kcat/Ks are independent of the puromycin concentration and the percentage of active ribosomes, as explained previously (24).

Effect of the Absence of L41 on Cycloheximide Resistance-- Cycloheximide is an inhibitor of polypeptide chain elongation. We examined the effect of cycloheximide on the growth of cells as well as on ribosome function in wild-type and mutant strains. The concentration of cycloheximide required for 50% inhibition of growth was 12 µg/liter for wild-type cells, 40 µg/liter for L41 mutant cells, 35 µg/liter for triple mutant cells, and 60 µg/liter for quintuple mutant cells.

Consistent with the above was the activity of wild-type and mutant ribosomes in the presence of cycloheximide. The concentration of cycloheximide required for 50% inhibition of polyphenylalanine synthesis was estimated at 0.14 µM for wild-type ribosomes, 0.40 µM for L41 mutant ribosomes, 0.44 µM for triple mutant ribosomes, and 0.81 µM for quintuple mutant ribosomes (Fig. 5). These results are in agreement with those reported for the inhibition of growth of the cells and show that the absence of L41 confers increased resistance to cycloheximide. The increased resistance to cycloheximide of triple mutant ribosomes, compared with wild type, is caused mainly by the absence of L24 (24).


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Fig. 5.   Inhibition of polyphenylalanine synthesis in the presence of cycloheximide. The extent of polyphenylalanine synthesis of each strain in the absence of antibiotic was taken as 100%. Plots: (black-square) wild type; (black-triangle) L41 mutant; (black-down-triangle ) triple mutant and () quintuple mutant ribosomes.

Translocation Studies-- Translocation of Ac-Phe-tRNA from the A- to P-site was monitored by reaction with 2 mM puromycin for 4 min at 30 °C. Elongation factor-independent (spontaneous) translocation was slow and proceeded in a linear fashion at least up to 8 min. Its rate was higher in the L41 mutant reaching 18% after 8 min compared with 10% in the wild type, if 100% indicates the extent of translocation in the presence of elongation factors. Therefore, ribosomal protein L41 suppresses spontaneous translocation.

In the presence of elongation factors and under conditions of single-round translocation, either elongation factor EF2 (0.1 µM) or the fraction containing the SPF promoted rapid translocation that was complete within 20 s in both strains as measured by the puromycin reaction (not shown). The puromycin reaction can detect changes in a reaction, which takes place in a time period of >= 5 s. No significant differences could be detected between the two strains either in the rate or the extent of enzymatic translocation.

The efficiency of translocation in the wild type and the L41 mutant was tested further by examining the dependence of each strain for translocation on the concentrations of EF2 (Fig. 6). To a fixed amount of pretranslocation complex, increasing concentrations of EF2 were added, and translocation was allowed to proceed for 1 min at 30 °C. Then puromycin was added and the reaction was terminated after 4 min. As shown in Fig. 6, the extent of translocation increased with EF2 concentration and reached a plateau as soon as peptidyl-tRNA was translocated on all active ribosomes in the reaction mixture. The concentrations of EF2 at which 50% translocation was achieved were 0.004 and 0.02 µM for the L41 mutant and the wild type, respectively. Interestingly, these results show that L41-lacking ribosomes are more efficient in enzymatic translocation than wild-type ribosomes.


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Fig. 6.   Dependence of translocation on elongation factor concentration. Pretranslocation Zwittergent 3-12 complexes were incubated with different amounts of EF2 for 1 min at 30 °C and the extent of translocation was determined by reaction with 2 mM puromycin for 4 min at 30 °C. The concentrations of EF2 at which 50% translocation was achieved were 0.004 µM for the L41 mutant and 0.02 µM for the wild type, respectively.

To test the inhibition of EF2-dependent translocation by cycloheximide, we added this antibiotic in ascending concentrations together with elongation factors for the specified time intervals and titrated the amount of Ac-Phe-tRNA translocated from the A- to P-site with the puromycin reaction (Fig. 7). Translocation was inhibited by 50% at 1.2 µM cycloheximide in the wild-type strain and at 2.5 µM cycloheximide in the L41 mutant strain. Consequently, the L41 mutant strain was shown to be more resistant to this antibiotic. These results are consistent with those reported earlier.


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Fig. 7.   Inhibition of EF2-dependent translocation in the presence of cycloheximide. The amount of Ac-[3H]Phe-tRNA translocated to the P-site was titrated with 2 mM puromycin for 4 min at 30 °C. The extent of translocation in the absence of antibiotic was taken as 100%. Plots: (black-square) wild type and (black-triangle) L41 mutant ribosomes.

Effect of the Absence of L41 on Polysome Profiles-- The effect of protein L41 in the assembly of the ribosome was examined through sucrose gradient analysis. The ratio of 60 S to 40 S ribosomal subunits remained stable, indicating that L41 does not participate in the maturation of the 60 S subunit. This conclusion is reinforced from the two-dimensional gel electrophoresis, which showed that L41 does not affect the incorporation of other ribosomal proteins in the 60 S subunit (Fig. 3). In contrast, the polysome profile of the L41 mutant strain indicated a significant decrease in the amount of 80 S ribosomes. These results imply that protein L41 is involved in ribosomal subunit association.

This possibility was confirmed by comparison of the polysome profiles of the quintuple and triple mutants. There was a further decrease in 80 S ribosomes from the quintuple mutant compared with those of the triple mutant, whereas the ratio of 60 S to 40 S subunits remained constant. The profiles of these two mutants also contained aberrations contributed by the absence of L24 and L39 (21). These aberrations included the appearance of half-mers, i.e. 43 S complexes consisting of the 40 S subunit with attached initiation factors, unable to bind defective 60 S. They also included a decrease in the 80 S monosomes caused by the absence of L24, as well as a decrease in the amount of 60 S caused by the absence of L39. Finally, a slight shift exists in the positions of the 80 S and 60 S peaks in both the triple and quintuple mutants, caused by the absence of these 60 S ribosomal subunit proteins.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In an effort to contribute to the understanding of yeast ribosomal structure and function, we examined the role(s) of ribosomal protein L41. For this purpose we used two strains, one in which the two genes for L41 were deleted and a second strain, in which the two genes for L41 were deleted from a background already lacking the genes for L24 and L39. We have found that in the absence of L24 and L39 the cell exhibited reduced protein synthesis activity and decreased translational accuracy, two closely related functions (21). Thus, this was an appropriate background in which to study the effect of the absence of a third protein, L41, on the above functions.

We found that protein L41 is dispensable for the yeast S. cerevisiae. This is the third case of a eukaryotic ribosomal protein, not found in eubacteria, which is nonessential for cell viability. The other two nonessential proteins, L24 and L39, were found to have significant effects on certain parameters of protein synthesis (21, 24). Specifically, L24 acted on the assembly of ribosomes and the kinetics of protein synthesis, whereas L39 acted on the accuracy of translation and the assembly of the 60 S subunits. Protein L41 is dispensable but it is not entirely innocuous to the cell: doubling times were somewhat longer in L41 mutants than wild type. Lack of L41 caused slight hyperaccuracy (Table II) accompanied by an increase in the resistance to paromomycin. Also, whereas the ratio 60:40 S remained stable, there was a decrease in the amount of 80 S ribosomes. Because this was not accompanied by a similar decrease in the amount of polysomes, it could imply that L41 is not involved in the initiation phase of protein synthesis, a notion that needs further evaluation.

The most pronounced effects of L41 are on the ribosomal peptidyltransferase activity (Table III and Fig. 4) and the translocation process of protein synthesis (Figs. 6 and 7). The peptidyltransferase activity of the ribosome was lowered 3-fold in the absence of L41. Likewise, the catalytic activity of the quintuple mutant was substantially lowered relative to that of the triple mutant. These results indicate a significant role for L41 in peptide bond formation. The presence of this dispensable protein is required so that the ribosome can exhibit its full catalytic activity. Because the ribosome is a ribozyme, it can only be surmised that L41 exerts its effect indirectly, possibly via allosteric interactions.

The absence of L41 allowed increased elongation factor-independent (spontaneous) translocation. Also, the absence of L41 apparently increased the efficiency of elongation factor-dependent translocation, as shown by the lower amounts of EF2 needed to achieve 50% translocation. These data indicate that L41 may belong to a class of ribosomal proteins with a distinct role, that of preventing translocation from occurring spontaneously.

Consistent with the role of L41 in translocation is the fact that its absence increased resistance to cycloheximide, a translocation inhibitor (Figs. 5 and 7). These results also suggest that yeast L41 participates in the binding site of cycloheximide on the ribosome. Mutations in two other ribosomal proteins, L42 (39) and L28 (40), were also shown to affect cycloheximide resistance. Subsequently, it was suggested that these two proteins may also play a central role in forming the cycloheximide binding site on the 60 S subunit (39).

Multiple deletion mutants are useful in the study of ribosomal assembly and function. A quintuple mutant carrying a deletion of the two genes encoding L41, the two genes encoding L24, and the single gene encoding L39 permitted the cells to remain viable and functioning. The fact that each one of the three proteins is not essential does not necessarily mean that the phenotype obtained from the five gene deletions should have been expected; the three deficiencies together might have resulted in a lethal phenotype. This mutant provided also an alternative way to study the functions of protein L41. This was achieved by comparing the changes that ribosomes from these quintuple mutant cells undergo to those from a triple mutant lacking only L24 and L39. The comparative study of these two mutants reaffirmed the earlier findings that L41 has a limited impact on cell growth, association of ribosomal subunits, translational accuracy, and resistance to paromomycin, but does not affect cell viability or polyphenylalanine synthesis.

A-site binding was significantly increased in the quintuple mutant compared with wild type (Table III). It has been suggested that an increase of A-site binding may arise from a higher affinity for accepting noncognate tRNAs and leads to a higher level of translational errors (41, 42). Because the quintuple mutant exhibited a high translational error rate (Table II), our results are in agreement with this suggestion. In contrast, lack of L41 affected A-site binding slightly. Thus, the quintuple mutant strain exhibited slightly lower A-site binding over the triple mutant and so did the L41 mutant over the wild type, in agreement with the fact that L41 causes slight hyperaccuracy.

It is interesting to note that the absence of L24 and L39 did not render the ribosome more susceptible to the absence of L41. In fact, the differences observed between L41 mutant and wild type were very similar to those between quintuple and triple mutants and fully accounted for by the absence of L41 alone in each of the two cases.

The quintuple mutant YKS121 provides not only a useful tool with which to investigate the role of ribosomal protein L41; it also provides a measure of the degree of deterioration of the vital activities a eukaryotic cell can withstand. It is shown that cells tolerate at least a 4-fold decrease in the rate of protein synthesis as measured by the puromycin reaction (Table III) combined with a 31/2-fold decrease in the fidelity of translation (Table II).

It is worth mentioning that polyphenylalanine synthesis is marginally affected in the L41 mutants over the wild type or in the quintuple over the triple mutants, whereas peptidyltransferase activity is lowered 3- and 2-fold, respectively, although peptide bond formation is a reaction of the elongation cycle of protein synthesis. A similar effect, i.e. a significant reduction of peptide bond formation but much less impairment of polyphenylalanine synthesis has been observed with a series of 23 S rRNA mutations (43) or with ribosomal protein L2 (23). These results may be explained by the hypothesis that the two processes have different rate-limiting steps. In fact the rate-limiting step of the elongation cycle is the occupation of the A-site and this is much slower than peptidyl transfer (44, 45). For the puromycin reaction, however, the rate-limiting step is the peptide bond formation and not the binding of puromycin to the A-site (23). As a result, a significant reduction in the rate of peptidyltransferase activity would more strongly decrease the rate of the puromycin reaction than the rate of polyphenylalanine synthesis.

In conclusion, the absence of protein L41 affected in varying degrees three of the main activities of the ribosome, i.e. peptide bond formation, translocation, and decoding, adding to the notion that these activities are interrelated and that some ribosomal proteins may possess more than one ribosomal function. Similar observations have been made recently for other ribosomal proteins, such as L39, the absence of which decreased translational fidelity but increased somewhat the ribosomal peptidyltransferase activity (21, 24). Likewise, several deleterious mutations in domain V of 23 S rRNA (9) have been linked with peptide bond formation whereas they also affect the fidelity of decoding, an activity of the small ribosomal subunit. Moreover, a model has been proposed in Escherichia coli whereby elongation factor G promotes translocation by modulating the communication between the peptidyltransferase domain of 23 S rRNA and the decoding region of 16 S rRNA during elongation (46). This communication may be achieved in various ways; for example, signals from the decoding center on the small subunit to the peptidyltransferase center on the large subunit can be transmitted either by ligands that contact both regions, e.g. bound tRNAs, or by the intersubunit bridges that connect the subunits (47).

    ACKNOWLEDGEMENTS

We thank Professor Jon Warner for making several yeast strains available to us. We also thank Dr. Irina L. Derkatch for providing strain 38C. We are indebted to Dr. Albert Sitikov for the generous gift of EF2. We also thank Dr. Theodora Choli-Papadopoulou for active interest and help and Dr. Dimitrios Kalpaxis for many helpful discussions and critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by a grant from the General Secretariat of Research and Technology, Ministry of Development of Greece (624/PENED01) to Dennis Synetos and Human Frontier Science Program Grant RG-360/93 (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a 3-year scholarship by the Greek State Foundation of Scholarships. Current address: Dept. of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637.

To whom correspondence should be addressed. Tel.: 302610-996125; Fax: 302610-997690; E-mail: dsynetos@med.upatras.gr.

Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M207533200

    ABBREVIATIONS

The abbreviations used are: Ac, acetyl; EF2, elongation factor 2; SPF, soluble protein factors.

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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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