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J. Biol. Chem., Vol. 278, Issue 34, 32236-32245, August 22, 2003
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¶
From the
Guelph-Waterloo Center for Graduate Work
in Chemistry and Biochemistry, Department of Chemistry and Biochemistry,
University of Guelph, Ontario N1G 2W1, the
Department of Cell Biology, University of
Alberta, Alberta T6G 2H7, Canada, and Institute for Systems Biology, Seattle,
Washington 98103
Received for publication, March 18, 2003 , and in revised form, June 5, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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mutants
defective in nuclear export. Furthermore, depletion of Utp8p blocked nuclear
export of mature tRNAs derived from both intronless and intron-containing
pre-tRNAs but did not affect tRNA and rRNA maturation, nuclear export of mRNA
and ribosomes, or nuclear tRNA aminoacylation. Overexpression of Utp8p also
alleviated nuclear retention of non-aminoacylated tRNATyr in a
tyrosyl-tRNA synthetase mutant strain. Utp8p binds tRNA directly and
saturably, indicating that it has a tRNA-binding site. Utp8p does not appear
to function as a tRNA export receptor, because it does not shuttle between the
nucleus and the cytoplasm. Taken together, the results suggest that Utp8p is
an essential intranuclear component of the nuclear tRNA export machinery,
which may channel tRNA to the various tRNA export pathways operating in S.
cerevisiae. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-karyopherin family
of nuclear import/export receptors, which translocate macromolecules through
the NPC by interacting with specific nucleoporins, and their function is
regulated by RanGTPase, a small Ras-like protein
(2,
3).
Nuclear tRNA export is facilitated by exportin-t and exportin-5 in
mammalian cells, and Los1p, the orthologue of exportin-t, in Saccharomyces
cerevisiae
(6–10).
These proteins are members of the -karyopherin family of
nucleocytoplasmic transport factors and bind the tRNA cargo directly in a
RanGTP-dependent manner in vitro
(6–8).
Exportin-t and exportin-5 are nucleoplasmic proteins, whereas Los1p is found
associated with the NPC (6,
8–11).
The function of Los1p is not essential, because disruption of the chromosomal
LOS1 gene did not affect growth or viability of S.
cerevisiae. This finding suggests that in addition to Los1p another
receptor is required for nuclear tRNA export in S. cerevisiae.
Early studies in Xenopus laevis suggest that aminoacylation of tRNAs in the nucleus plays a role in nuclear tRNA export (12). This conclusion is based on the observation that tRNATyr or tRNAMet microinjected in the nucleus of X. laevis is aminoacylated, and loss of nuclear aminoacylation of tRNATyr in oocytes by inhibition of the tyrosyl-tRNA synthetase (TyrRS) (12) led to a significant decrease in the efficiency of nuclear export of the RNA. Moreover, aminoacyl-tRNA synthetases have been detected in the nucleus of mammalian cells (13). Mutants of tRNAPhe that are defective in aminoacylation, however, were found to be exported to the cytoplasm after injection into the nucleus of X. laevis oocytes (14). This finding suggests that nuclear tRNA aminoacylation is not absolutely required for tRNA export in mammalian cells. Aminoacylated tRNAs were also detected in the nucleus of an S. cerevisiae nup116 mutant strain defective in nuclear export (15). In addition, nuclear retention of tRNA was observed in several aminoacyl-tRNA synthetase mutant strains and in wild type strains when aminoacylation was blocked by amino acid starvation (16, 17). Subcellular fractionation detected TyrRS in the nucleus of S. cerevisiae (18). In addition, the enzyme was shown to contain a nuclear localization signal (18). Mutation of the nuclear localization signal caused a reduction in the nuclear pool of the protein as well as a block in nuclear export of tRNATyr (18). However, this mutation did not affect the aminoacylation activity of the enzyme or the viability of the cells (18). These results provided very good evidence that nuclear aminoacylation also plays a role in nuclear tRNA export in S. cerevisiae, but it is not absolutely required. Nuclear tRNA aminoacylation may constitute a Los1p-independent export pathway, because overexpression of the methionyl-tRNA synthetase restored export of tRNAMet but not tRNAIle in the los1 mutant strain (19). The receptor that facilitates nuclear export of aminoacylated tRNAs in S. cerevisiae is not known.
The ATP (CTP):nucleotidyltransferase (Cca1p) is an essential enzyme that prepares tRNAs for aminoacylation in the nucleus, cytoplasm, and mitochondrion by adding the nucleotides C, C, and A to the 3' ends of tRNAs. This maturation step, but not aminoacylation itself, appears to be absolutely required for nuclear export of tRNAs in both mammalian cells and S. cerevisiae, and because it has been shown that exportin-t preferentially binds tRNAs with the 3' CCA ends, nuclear tRNA export is blocked in a cca1 S. cerevisiae mutant strain, and tRNAs lacking CCA were not exported to the cytoplasm in X. laevis (12, 14, 16, 17, 20). Recent studies also indicated that Cca1p is directly involved in nuclear tRNA export in S. cerevisiae. Like methionyl-tRNA synthetase, overexpression of Cca1p restored nuclear export of tRNAMet, a tRNA made from intronless pre-tRNA, in the los1 mutant strain, and the protein was shown to shuttle between the nucleus and cytoplasm (19). These results led to the suggestion that Cca1p may function as a tRNA export receptor or an adaptor in a Los1p- and nuclear aminoacylation-independent pathway that is required for export of tRNAs obtained from intronless pre-tRNAs (19). This is consistent with the finding that loss of Los1p function and nuclear tRNA aminoacylation did not affect the viability of the cells (18). However, it is not known whether Cca1p or another unidentified Los1p- and aminoacylation-independent pathway facilitates nuclear export of tRNAs derived from intron-containing pre-tRNAs.
The genetic and biochemical studies reported suggest that nuclear tRNA export in S. cerevisiae involves multiple redundant pathways. The details of these pathways, however, are poorly understood, and only a small number of the proteins that participate in nuclear tRNA export are known. We reported previously (21) the development of an amber suppression phenotypic assay in S. cerevisiae to identify eukaryotic proteins associated with the nuclear tRNA export process. This method involves the use of nuclear export-defective mutants of the yeast tyrosine amber suppressor tRNA as reporters to identify genes of proteins that can restore their export. In this report we employ a yeast tRNA three-hybrid interaction approach and the amber suppression assay to identify proteins of the nuclear tRNA export apparatus of S. cerevisiae. This strategy resulted in the identification of several proteins including Utp8p, an essential 80-kDa nucleolar protein that has been implicated in 18 S ribosomal RNA (rRNA) biogenesis (22). Our genetic and biochemical characterizations showed that the primary function of Utp8p is in nuclear tRNA export. The data suggest that the protein acts at a step in-between tRNA maturation/aminoacylation and translocation of the tRNA out of the nucleus. Utp8p may serve as an intranuclear factor that delivers aminoacylated and non-aminoacylated tRNAs to the appropriate tRNA export pathway.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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plasmid containing the URA3 selection marker was obtained from Dr. P.
Good, Department of Biological Chemistry, University of Michigan
(24). The pRS416-CEN-URA3
vector was purchased from Stratagene, and pRS423 was obtained from the
American Type Culture Collection. The pRS313-Xpo1-GFP plasmid (pKW470) was
obtained from Dr. Karsten Weis, Department of Molecular and Cell Biology,
University of California, Berkeley, and the plasmids pRS TYS1-nls1-myc and
pRSTYS1-myc were provided by Dr. A. K. Hopper, Department of Biochemistry and
Molecular Biology, Pennsylvania State University College of Medicine
(18,
25). The 2-µm yEPLAC195
plasmid carrying the synthetic yeast
gene lacking
the intron found in the natural gene and pYX242 carrying the LOS1
gene were described before
(21). The plasmids
pRS-CEN-LEU2-RPL3-GFP and pRS-CEN-LEU2-RPL25-GFP were described previously
(26,
27). pIII-tRNA-MS2 was made by
ligation of an EcoRI fragment containing the MS2 RNA gene and a small
linker region from pIII-MS2-1 into the same site in
.
pYX242-UTP8 was constructed by introducing an EcoRI-BamHI
fragment containing the ORF of Utp8p into the same sites in pYX242; the Utp8p
ORF was prepared by PCR using S. cerevisiae chromosomal DNA. In the
pYX242 vector, the UTP8 gene is under the control of the
triose-phosphate isomerase promoter and transcription-termination sequence.
pET(His)6-UTP8 was constructed by inserting an
NdeI-BamHI fragment of the Utp8p ORF into the same sites in
pET19b His6; the fragment containing the Utp8p ORF was prepared by
PCR using the pYX242-UTP8 vector as the template. pCEN-URA-GAL1-UTP8 was
constructed by cloning an EcoRI-SmaI fragment containing the
UTP8 gene into the EcoRI and SalI (blunted) sites
in the pRS416-CEN-URA-GAL1 vector. pCEN-URA-GAL1-XPO1-GFP was made by ligating
an SpeI-SmaI fragment containing the Xpo1-GFP gene into the
same sites in pCEN-URA-GAL1; the fragment was obtained by PCR using the
pRS313-Xpo1-GFP plasmid as the template. pCEN-URA-GAL1-UTP8-GFP was obtained
by cloning an EcoRI-SalI fragment containing the Utp8-GFP
gene into the EcoRI-XbaI sites in pCEN-URA-GAL1 vector;
incompatible ends were filled in using Klenow. pRS423-UTP8 was prepared by
ligating a SacI fragment containing the entire UTP8 gene
into the same site in pRS423; UTP8 was obtained by PCR using S.
cerevisiae chromosomal DNA as the template. The pRS423-CCA1 plasmid was
constructed by cloning a BamHI fragment containing the entire
CCA1 gene into the same site in pRS423, the CCA1 gene was
prepared by PCR using S. cerevisiae chromosomal DNA as the template.
The 
ACT2 S. cerevisiae cDNA library was purchased from the
American Type Culture Collection and converted to plasmids (pACT2 carrying a
LEU2 selection marker) using Escherichia coli BNN132
(28). The S.
cerevisiae strains used in this study are listed in
Table I.
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tRNA Three-hybrid Screen of an S. cerevisiae cDNA Library for Genes of Proteins That Interact with tRNA—L40coat harboring the pIII-tRNA-MS2 vector was transformed with the S. cerevisiae cDNA library in pACT2 (29). The transformed cells were plated on complete synthetic dextrose (CSD) medium lacking uracil, leucine, and histidine (CSD-Ura-Leu-His) and containing 10 mM 3-aminotriazole. Transformants appeared within 7 days of incubation at 30 °C. The His+ transformants were tested for lacZ expression using the colony lift assay as specified by Clontech.
Isolation of the pACT2 Library Plasmid from
His+LacZ+
L40coat Transformants—The
transformants were grown at 30 °C on CSD-Leu medium to select for the pACT
plasmid. A single colony was streaked on CSD-Leu medium containing 0.1%
5-fluoroorotic acid to select for cells lacking the pIII vector carrying the
tRNA-MS2 fusion gene. The pACT2 plasmid was isolated from the
L40coat transformant and amplified in DH5
. DNA sequencing
followed by a BLAST search of the S. cerevisiae genome data base
provided the complete DNA sequence and identity of the cloned genes.
Amber Suppression Analysis of the Effect of Overproduction of Utp8p on
Nuclear Export of 
Mutants Defective in
Export in S. cerevisiae—The yEPLAC195 plasmid with and without the
gene for the wild type
or the
G11:C24
mutant was electroporated into a HEY301-129 transformant carrying pYX242-UTP8
or pYX242-LOS1, and transformants were selected on CSD-Leu-Ura medium. The
transformants were grown at 30 °C in CSD-Leu-Ura medium and suppression of
amber codons in the trp1 gene in HEY301-129 was assessed by growth of
the transformants on CSD-Leu-Ura-Trp
(21).
Isolation of a Conditional utp8p Mutant Strain—The BY4743 (UTP8/utp8::KANR) heterozygote harboring pCEN-URA-GAL1-UTP8 was sporulated, and tetrads were dissected on YP medium containing 2% raffinose. The haploids were screened for Ura+ G418R on CS medium lacking Ura and containing 2% raffinose, 2% galactose, and 200 µg/ml G418.
Fluorescence in Situ Hybridization (FISH) Analysis of the Nucleocytoplasmic Distribution of tRNAs—The utp8 strain (BYU8) carrying the pCEN-URA-GAL1-UTP8 plasmid was grown overnight at 30 °C in CS medium lacking Ura and containing 2% raffinose and 200 µg/ml G418. The cells were diluted to an A600 of 0.1 in CS medium lacking Ura and containing 2% raffinose and 2% glucose or 2% galactose and grown at 30 °C for 6 h. These conditions were also used for growth of HEY301-129 transformants except CSD-Leu-Ura medium was used. The cells (3 ml of culture) were treated as described (15, 17) and incubated at 37 °C for 2 h in hybridization buffer (4x SSC, 50% formamide, 10% dextran sulfate, 125 µg/ml E. coli 5 S rRNA, 500 µg/ml salmon sperm DNA, 0.5 units/µl RNasin (Promega), 1x Denhardt's). Hybridization was carried out at 37 °C for 12 h in hybridization buffer containing 0.5 pmol/µl of 5'-end fluorescein-labeled oligonucleotide. The cells were washed two times (10 min each at 45 °C) with 2x SSC and three times (10 min each at room temperature) with 1x SSC. 4',6'-Diamidoindo-2-phenylindole (1 µg/ml) was used to visualize nuclear DNA. The slides were viewed under a 60x objective lens of a Nikon Eclipse 6600 microscope. The images were recorded using a Coolsnapfx monochrome CCD digital camera (Roper Scientific) and processed using Metamorph (Universal Imaging).
Fluorescent Oligonucleotides—5'-End
fluorescein-labeled oligonucleotides were obtained from Invitrogen. The
and
tRNATyr were detected with 5'-CAAGATTTAGAGTCTTG-3' and
5'-CAAGATTTACAGTCTTG-3', respectively.
5'-GGCCCAACGATGGCAACG-3' was used to detect tRNAGly.
These oligonucleotides are complementary to sequences in the anticodon
stem-loop of the mature tRNAs. 5-GGTCTTACTTCCCATC-3' was used to detect
the U18 small nucleolar RNA (U18 snoRNA).
Overproduction and Purification of Utp8p Containing an N-terminal
His6 Tag—The His6-tagged
Utp8p was overproduced in E. coli BL21 Codon Plus (Novagen). A
transformant carrying the pET(His)6-UTP8 vector was grown overnight
at 37 °C in Luria-Bertani medium containing 100 µg/ml ampicillin and 30
µg/ml chloramphenicol. The culture was diluted 50-fold into 2 liters of 2YT
containing the antibiotics and grown at 37 °C until the culture reached an
A600 of 0.6. Expression of the His6-tagged
Utp8p was induced for 20 min using 0.02 mM
isopropyl-1-thio--D-galactopyranoside. The cells were
pelleted by centrifugation and resuspended in 40 ml of 20 mM
Tris-HCl, pH 7.5, buffer containing 15 mM imidazole, 100
mM NaCl, and a mixture of protease inhibitors (Roche Applied
Science) (binding buffer). The cells were lysed at 70,000 kPa using a French
press, and unlysed cells and debris were removed by centrifugation at 10,000
x g for 10 min at 4 °C
(30). The supernatant was
applied to a Talon Co2+ affinity column (Clontech) (2-ml
bed volume) pre-equilibrated with binding buffer. The column was washed with
20 ml of binding buffer, followed by 20 ml of binding buffer containing 50
mM imidazole. The bound protein was eluted from the column with
binding buffer containing 300 mM imidazole and dialyzed against 20
mM HEPES buffer, pH 7.6, containing 150 mM NaCl at 4
°C. Rabbit antibodies against purified Utp8p were prepared by ResGen,
Huntsville, AL.
Western Blot Analysis of Utp8p Expression—The utp8
mutant strain harboring the pCEN-URA-GAL1-UTP8 vector was grown overnight at
30 °C in CS medium lacking Ura and containing 2% raffinose and 200
µg/ml G418. An aliquot of the culture was diluted to an
A600 of 0.1 in CS medium lacking Ura and containing 2%
raffinose and 2% glucose or 2% galactose and grown at 30 °C. At the
required times, an aliquot of the culture corresponding to the same number of
cells, based on A600, was pelleted and washed with water.
The cells were lysed in 7.4% -mercaptoethanol and 1.85 N NaOH
as described (27), and the
proteins were precipitated with 10% trichloroacetic acid. The protein
precipitate was rinsed with water and solubilized by boiling for 5 min in 62.5
mM Tris-HCl, pH 6.8, buffer containing 5% SDS (w/v), 10% glycerol
(v/v), and 0.02% bromphenol (v/v). The proteins were separated on a 10% PAGE
and transferred electrophoretically to Protran nitrocellulose membrane. Utp8p
was detected with a rabbit anti-Utp8p antibody using the ECL detection system
(Amersham Biosciences).
Northern Blot Analysis of the State of tRNA Processing and Maturation—The utp8 and UTP8 (BYU) strains carrying the pCEN-URA-GAL1-UTP8 plasmid were grown overnight at 30 °C in CS medium lacking Ura and containing 2% raffinose and 200 µg/ml G418. The cells were diluted to an A600 of 0.1 in CS medium lacking Ura and containing 2% raffinose and 2% glucose or 2% galactose and grown at 30 °C for 6 h. The PUS1 and pus1 strains were grown in YPAD containing 200 µg/ml G418 as described above. The los1 strain was grown in CSD-His medium. Total RNA was isolated from the various strains and separated on a 10% polyacrylamide gel containing 8 M urea using 1x TBE at room temperature (21) or on a 6.5% polyacrylamide gel containing 8 M urea at 4 °C using 100 mM sodium acetate buffer, pH 5.0 (21, 31–34). The separated RNAs were transferred electrophoretically onto Nytran membrane. The membranes were incubated at 37 °C for 4 h in prehybridization solution consisting of 4x SSPE (1x SSPE = 0.18 M NaCl, 10 mM NaH2PO4, 1 mM Na2EDTA), 250 µg/ml sheared and denatured salmon sperm DNA, 0.1% SDS, and 10x Denhardt's solution (1x Denhardt's = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone 40, and 0.02% Ficoll). Hybridization was performed at 37 °C overnight in pre-hybridization solution containing 5'-end 32P-labeled oligonucleotide (1–2 x 106 cpm/ml). The membranes were washed twice for 30 min at room temperature and once for 30 min at 38 °C with 1x SSPE and 0.1% SDS and subjected to autoradiography.
Analysis of the State of tRNA Aminoacylation in the
Nucleus—The utp8 strain carrying the pCEN-URA-GAL1-UTP8
plasmid was grown overnight at 30 °C in CS medium lacking Ura and
containing 2% raffinose and 200 µg/ml G418. The cells were diluted to an
A600 of 0.1 in CS medium lacking Ura and containing 2%
raffinose and 2% glucose or 2% galactose and grown at 30 °C for 6 h. The
nuclear and post-nuclear fractions were isolated as described
(21). The cells were washed
with 40 ml of 0.5% -mercaptoethanol and resuspended in 40 ml of SB (1.2
M sorbitol, 10 mM EDTA, pH 8.0, 10 mM
KPO4, pH 7.5, 0.1% -mercaptoethanol). The cells were
converted to spheroplasts by incubating the suspension with 5 mg of Zymolyase
100T at 30 °C with gentle agitation. The incubation mixture was
centrifuged at 2500 x g for 1 min. All subsequent steps were
performed at 4 °C. The cells were washed with 15 ml of AMC (300
mM sodium acetate, pH 5.0, 5 mM magnesium acetate, 0.5
M sucrose) and resuspended in 25 ml of AMS (300 mM
sodium acetate, pH 5.0, 5 mM magnesium acetate, 0.25 M
sucrose) with 0.1% Nonidet P-40. The cells were lysed by homogenization with a
Dounce homogenizer, using 25 strokes with a loose pestle followed by 10
strokes with a tight pestle. The unlysed cells were removed by centrifugation
at 2500 x g for 10 min. The supernatant was applied to an 8-ml
AMC cushion and centrifuged at 8000 x g for 10 min. The
resulting nuclear (pellet) and post-nuclear (supernatant) fractions were
separated, and the pellet was resuspended in 100 µl of AMS. An equal volume
of phenol was added to the nuclear and post-nuclear fractions, and the mixture
was vortexed every 2 min for 30 s. The mixture was centrifuged, and the
aqueous phase was extracted with an equal volume of phenol:chloroform,
followed by a final extraction with an equal volume of chloroform. To the
aqueous phase was added 3 volumes of 95% ethanol. The mixture was incubated at
–20 °C overnight and centrifuged at 5000 x g for 30
min. The RNA precipitate was resuspended in an appropriate volume of 20
mM sodium acetate, pH 5.0. Total RNA from the nuclear and
post-nuclear fractions was separated by electrophoresis on a 6.5%
polyacrylamide gel containing 8 M urea at 4 °C using 100
mM sodium acetate buffer, pH 5.0, and transferred onto Nytran Plus
membranes. Northern analysis was performed as described above. Deacylated tRNA
marker was prepared by incubating nuclear and cytoplasmic RNA in 50
mM Tris-HCl, pH 9.0, at 37 °C for 1 h.
Purification of E. coli 5 S rRNA—Total RNA was isolated from
E. coli as described
(32). A 500-µl aliquot of
the RNA was mixed with an equal volume of loading buffer (8 M urea,
0.05% bromphenol blue, 0.05% xylene cyanol in 1x TBE) and subjected to
electrophoresis on an 8% polyacrylamide gel containing 8 M urea
(700 V, 
5 h). The RNA bands were visualized by UV shadowing, and the band
corresponding to the 5 S rRNA was excised. The RNA was extracted from the gel
with TE, pH 8.0, by shaking at 30 °C. The RNA was precipitated and
dissolved in TE, pH 8.0.
Utp8p-RNA Interaction—Substrate-induced intrinsic fluorescence quenching was used to determine whether Utp8p binds tRNA. The reaction mixtures contain 20 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.25 µM Utp8p, and a varying amount of a mixture of mature yeast tRNA (Sigma) or E. coli 5 S rRNA (0.25, 0.50, 1, 2, 4, 6.25, 8, 12.5, 16, and 18.75 µM) and incubated for 1 h at 4 °C. Control reactions containing tRNA alone were prepared as above. Trp and Tyr fluorescence was measured using a Photon Technology International spectrofluorimeter (London, Ontario, Canada) with excitation and emission slits set to 4 nm, and excitation and emission wavelengths of 280 and 318 nm, respectively (30). The fluorescence intensity of each sample was subtracted from that of the appropriate control reaction and expressed as a percent reduction of the fluorescence intensity obtained with Utp8p alone.
Heterokaryon Shuttling Assay—W303 (MATa) strain
harboring pCEN-URA-GAL1-UTP8-GFP or pCEN-URA-GAL1-XPO1-GFP was grown in CS
containing 2% raffinose and 2% galactose and lacking Ura to mid-logarithmic
phase and then in selective medium containing glucose for 1 h. The donor cells
were mixed with the recipient kar1-1 (MAT) strain
grown in CSD medium, and the cells were pelleted by centrifugation at room
temperature (26). The cell
pellet was resuspended in CSD medium and incubated at room temperature. After
30 min an aliquot of the cell suspension was placed on a microscope slide
coated with CSD medium containing 2% agarose. A coverslip was placed over the
sample and sealed. The slide was incubated at room temperature for 5 h and
then subjected to direct fluorescence microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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gene
lacking the intron sequence fused to the 5' end of the MS2 RNA gene. The
RNA polymerase III promoter of the tRNA gene directs transcription of the
hybrid RNA gene. Synthesis of the
hybrid RNA was confirmed by Northern blot analysis (data not shown). The
L40coat strain harboring the pIII-tRNA-MS2 plasmid and a
chromosomal copy of the LexA DNA-binding domain-MS2 coat protein hybrid gene
was transformed with a library of S. cerevisiae cDNAs fused to the
GAL4 activation domain and selected for activation of expression of
the HIS3 reporter gene (Fig.
1). Of the 1.5 million transformants screened, 125 colonies
expressed both the HIS3 and lacZ reporter genes. Many of the
genes isolated by this strategy encode known RNA-binding proteins, including
the tRNA-binding proteins eukaryotic elongation factor eEF-1A, GCN2, and the
La protein (35,
36), as well as several genes
encoding proteins of unknown function. In addition, the gene YGR128c
encoding Utp8p was also isolated. Utp8p is an essential 80-kDa nucleolar
protein that is thought to play a role in 18 S rRNA biogenesis
(22). Utp8p does not have any
significant identity or similarity to any known proteins, including those
known to be involved in nuclear tRNA export. However, findings presented below
showed that the major function of Utp8p is in nuclear export of tRNA.
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Utp8p Restored Export of Mutants
Defective in Nuclear Export—We have shown previously
(21) by amber suppression and
nuclear cytoplasmic distribution analyses that the G11:C24
mutant is
defective in nuclear export in S. cerevisiae. This defect is not due
to lack of maturation of the tRNA and can be rescued by overexpression of
Los1p or exportin-t but not by Arc1p, the Tfc5p subunit of transcription
factor TFIIIB, or elongation factor eEF3
(21). To ascertain whether
Utp8p could be involved in nuclear tRNA export, the effect of overexpression
of Utp8p on nuclear export of the G11:C24
mutant was
investigated. Export of the G11:C24
mutant was
detected by growth of the transformants on Trp amber medium, which selects for
amber suppression (Fig.
2A). Transformants harboring the pYX242 vector without
(sector 1) or with the UTP8 (sector 4) or LOS1 (sector 7)
gene and lacking the
were unable
to grow on the Trp amber medium. As expected, cells expressing the wild type
alone
(sector 2) or with Utp8p (sector 5) or Los1p (sector 8) grew on the Trp amber
medium. In contrast, no amber suppression was observed for the transformant
expressing the G11:C24 mutant
alone
(sector 3). However, amber suppression was observed when the G11:C24
mutant and
Utp8p (sector 6) or Los1p (sector 9) were co-expressed.
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FISH was used to determine whether overproduction of Utp8p would also
facilitate export of the G11
mutant,
which is primarily retained in the nucleus
(21). Export of this tRNA
mutant is also rescued by overexpression of Los1p
(21). However, the G11
mutant does
not suppress amber codons due to an additional defect, which prevents the tRNA
from participating in protein synthesis
(21). The G11
mutant was
detected with a 5'-fluorescein-labeled oligonucleotide complementary to
nucleotides 29–47 of the anticodon stem and loop of
. We have
shown previously (21) by
Northern blot analysis that this probe is specific for the
, and FISH
analysis indicated that it did not hybridize to any significant extent to the
endogenous tRNATyr in vivo (data not shown). The
endogenous U18 snoRNA was used as a nuclear marker
(Fig. 2B)
(21). The wild type
was found in
the nucleus and cytoplasm of the cells. In transformants expressing the G11
mutant
alone, the tRNA was found predominantly in the nucleus, as reported previously
(Fig. 2B)
(21). In contrast,
overproduction of Utp8p or Los1p shifted accumulation of the G11
mutant
toward the cytoplasm (Fig.
2B). Taken together, these results suggest that Utp8p
plays a role in nuclear tRNA export in S. cerevisiae.
Depletion of Utp8p Blocked Nuclear Export of tRNAs Derived from Intronless and Intron-containing Pre-tRNA but Not mRNA and Ribosome Export or Ribosomal RNA and tRNA Maturation—Previous studies (37) have shown that disruption of the YGR128c ORF encoding Utp8p is lethal. To investigate whether depletion of Utp8p caused a block in nuclear tRNA export, a conditional utp8 mutant strain was prepared by tetrad dissection of a heterozygote harboring the pCEN-URA vector containing the UTP8 gene under the control of the inducible GAL1 promoter. The utp8 strain grew when expression of Utp8p was induced by galactose (Fig. 3A). However, when expression of Utp8p was repressed by glucose, the mutant strain grew very poorly (Fig. 3A). The use of 5-fluoroorotic acid to select for cells lacking the pCEN-URA-GAL1-UTP8 vector resulted in no cell growth, even after 6 days of incubation at 30 °C (data not shown). These results confirm that the function of Utp8p is essential. The growth observed for the utp8 strain on glucose is most likely due to a low level of expression of the Utp8 protein, because Western blot analysis detected a small amount of Utp8p when the mutant strain was grown for various times in medium containing glucose (Fig. 3B, a–c).
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To investigate whether tRNA is retained in the nucleus of the utp8 mutant strain, FISH was used to assess the cellular location of the endogenous tRNATyr, a tRNA derived from an intron-containing precursor, and of tRNAGly, which is made from a pre-tRNA lacking an intron. The utp8 mutant strain harboring the pCEN-URA-GAL1-UTP8 vector was first grown in medium containing raffinose and then for 6 h in medium containing raffinose and glucose or galactose. Western blot analysis showed that galactose induction increased the amount Utp8p dramatically (Fig. 3B, upper panel, compare lanes a and b). In the galactose-induced cells both tRNATyr and tRNAGly were detected in the cytoplasm (Fig. 3C). A low amount of Utp8p was observed after 6 h of glucose repression (Fig. 3B, upper panel, compare lanes b and c). This amount of Utp8p did not change even after an 8-h incubation of the cells in medium containing glucose (data not shown). The level of Utp8p after 6 h of depletion was considerably lower than that of the endogenous protein in the wild type UTP8 strain (Fig. 3B, lower panel). FISH analysis showed that both tRNATyr and tRNAGly were predominantly in the nucleus of glucose-repressed cells (Fig. 3C). In contrast, the cellular distribution of mRNA was not affected by the loss of Utp8p function (Fig. 4A). Furthermore, the large ribosomal subunits (rpL3 and rpL25) tagged with GFP were detected in the cytoplasm of the utp8 strain when Utp8p expression was repressed for 6 h (Fig. 4B, lower panel). The same localization pattern was observed prior to glucose repression and after galactose induction of Utp8p expression (Fig. 4B, upper panel). Although the overall levels of the proteins decreased, only a modest amount of rpL3 and rpL25 was observed in the nucleus when depletion of Utp8p was carried out for 24 h (Fig. 4B, lower panel). In addition, the levels of 25 S, 18 S, and 5 S rRNAs did not change significantly during the 24-h depletion period (Fig. 4C, left panel). Northern blot analyses also showed that the level of the 25 S and 18 S rRNAs did not decrease significantly during the 24-h period (Fig. 4C, right panel). The decrease observed for both rRNA at 12 h is most likely due to RNA degradation.
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To exclude the possibility that nuclear retention of the tRNAs was due to a defect in tRNA maturation, the processing and modification status of tRNA was investigated. tRNA processing was assessed in the UTP8 and utp8 strains harboring the pCEN-URA-GAL1-UTP8 vector by using Northern blot analysis to monitor the state of maturation of the endogenous tRNATrp. The strains were grown for 6 h at 30 °C in minimal medium containing raffinose and glucose or galactose, before total RNA was isolated. The oligonucleotide probe used to detect the various processed forms of tRNATrp is complementary to nucleotides of the T-stem-loop (38). As reported, the probe is capable of detecting mature tRNATrp and the three unspliced forms of the tRNA in the los1 strain, which is partly defective in tRNA splicing (38–40) (Fig. 5A, left panel). However, only the mature form of tRNATrp was detected in the Utp8p wild type (Fig. 5A, right panel, lanes 1 and 2) and mutant (lanes 3 and 4) strains irrespective of whether expression of the UTP8 gene was repressed (lanes 1 and 3) or induced (lanes 2 and 4).
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Previous studies (32,
33) have shown that an E.
coli initiator tRNA mutant or the E. coli tRNATyr
lacking a
2-methylthio-N6-(
2-isopentenyl)
modification can be easily separated from the fully modified tRNA using PAGE
at pH 5.0. Therefore, this approach was also used to investigate whether Utp8p
may play a role in tRNA modification. To verify that this method can
discriminate between modified and unmodified yeast tRNA, we compared the
electrophoretic behavior of the minor tRNAIle(UAU) present in total
RNA isolated from wild type and mutant Pus1p S. cerevisiae strains
(Fig. 5B, left
panel). Pus1p is an intron-dependent tRNA pseudouridine synthetase, which
has been shown to convert U34, U35, U36, and U27 of the minor
intron-containing tRNAIle (UAU) to pseudouridine
(11,
41,
42). This modification results
in CH at position 3 changed to NH. tRNAIle (UAU) from the
PUS1 strain (lane 1) migrated slightly faster than the tRNA
from the pus1 mutant (lane 2), showing that this
electrophoretic system can separate unmodified from modified yeast tRNAs. The
increased mobility of the fully modified tRNA is due to the amino group at
position 3 in pseudouridine (pKa = 7.5) being
positively charged under the acidic conditions used.
The most frequently found modification in tRNAs is conversion of uridine to pseudouridine. To determine whether pseudouridinylation of tRNA was affected in the utp8 strain, the electrophoretic system described above was used to assess the state of modification of tRNATrp (Fig. 5B, right panel). A single form of tRNATrp was observed in the UTP8 strain (Fig. 5B, lanes 1 and 2). This form of tRNATrp was detected in the utp8 mutant strain when expression of the UTP8 gene under the control of the GAL1 promoter in pCEN-URA was repressed (lane 3) or induced (lane 4). This result showed that loss of Utp8p function did not affect modification of uridine in tRNATrp to pseudouridine and implies that Utp8p is not playing a role in tRNA modification. However, we cannot exclude the possibility that another type of tRNA modification is affected, because it is not known whether other types of modification can influence the mobility of tRNA during electrophoresis under acidic conditions. Nonetheless, the notion that Utp8p is not required for tRNA maturation is consistent with the observation that nuclear tRNA aminoacylation was not affected (discussed below).
Loss of Utp8p Function Did Not Affect Nuclear tRNA
Aminoacylation—Nuclear tRNA aminoacylation is a requirement for
certain tRNA export pathways in S. cerevisiae. Therefore, the effect
of depletion of Utp8p on nuclear tRNA aminoacylation was investigated by
Northern blot analysis of the aminoacylation status of tRNATyr and
tRNAGly in total RNA obtained from nuclei prepared from the
utp8 strain when expression of UTP8 was induced and
repressed (Fig. 6). The acidic
conditions used to isolate tRNAs from nuclear and post-nuclear fractions, and
subsequent separation by PAGE, prevents hydrolysis of the ester bond linking
the amino acid to the tRNA. tRNATyr from the nuclear (left
panel, lane 3) and cytosolic (lane 2) fractions obtained from
the utp8 strain expressing the wild type Utp8 protein is primarily in
the aminoacylated form. Deacylation of the tRNAs (lanes 1 and
4) by treatment with base prior to electrophoresis resulted in a
faster migrating species corresponding to deacylated tRNATyr,
verifying that tRNATyr in both fractions was aminoacylated. Both
tRNATyr (middle panel, lanes 6 and 7) and
tRNAGly (right panel, lanes 9 and 10) were
present in the aminoacylated form in the nuclear (lanes 7 and
10) and cytosolic fractions (lanes 6 and 9)
isolated from the utp8 strain depleted of Utp8p. These results showed
that loss of Utp8p function did not significantly affect nuclear
aminoacylation of tRNAs derived from intronless (tRNAGly) and
intron-containing (tRNATyr) pre-tRNAs. In addition, they suggest
that depletion of Utp8p did not affect tRNA maturation. The amount of
tRNATyr in the cytoplasm was considerably lower than that in the
nucleus when Utp8p expression was turned off (compare lanes 6 and
7). A significant reduction in the level of tRNAGly in the
cytoplasm was also observed after a short exposure time. Quantification of
Northern blots by PhosphorImager analyses indicated that the level of nuclear
tRNA after depletion of Utp8p is 4–5 times higher compared with
that before repression of Utp8p expression (data not shown). These findings
confirm that loss of Utp8p function caused a block in tRNA export from the
nucleus.
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Utp8p Increased the Efficiency of Nuclear Export of tRNATyr in a TyrRS Mutant Strain—TyrRS has been shown to contain a nuclear localization signal (18). Mutation of this signal reduced the nuclear level of TyrRS but did not affect the catalytic activity of the enzyme. A yeast strain (ts2) harboring a chromosomal temperature-sensitive tys1 allele and expressing the mutant TyrRS protein (TYS1-nls1) was viable at 37 °C, because non-aminoacylated tRNATyr is exported from the nucleus by another pathway (18). However, the efficiency of export of tRNATyr is sufficiently reduced that FISH could detect accumulation of the RNA in the nucleus of the mutant strain (18). Therefore, to obtain further proof that Utp8p is involved in nuclear tRNA export, FISH was used to investigate whether overexpression of Utp8p would improve the efficiency of nuclear export of tRNATyr in the ts2 TYS-nls1 strain at 37 °C (Fig. 7A). Consistent with previous studies, nuclear retention of tRNATyr was observed in the ts2 TYS-nls1 strain. Retention of the tRNA was overcome by overexpression of wild type TyrRS or Utp8p but not by Cca1p.
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To verify that overexpression of Utp8p facilitates nuclear export of tRNATyr, Northern blot analysis was used to investigate the level of tRNATyr in the nuclear and cytosolic fractions prepared from the ts2 TYS-nls1, ts2 TYS, ts2 TYS-nls1 Utp8p, and ts2 TYS-nls1 Cca1p strains grown at 37 °C (Fig. 7B). The nuclear-cytoplasmic distribution analysis also detected accumulation of tRNATyr in the nucleus of the ts2 TYS-nls1 strain and the ts2 TYS-nls1 strain overproducing Cca1p. We have found that the portion of the cellular tRNATyr in the nuclear fraction from the ts2 TYS-nls1 strain varies from 50 to 70% (data not shown). In contrast to the ts2 TYS-nls1 strain, the majority of tRNATyr was found in the cytosolic fraction of the ts2 TYS and ts2 TYS-nls1 Utp8p strains. The percentage of the cellular tRNATyr in the nuclear fraction from the ts2 TYS and ts2 TYS-nls1 Utp8p strains varies from 10 to 30% (data not shown). Thus, these findings support the conclusion that Utp8p is involved in nuclear tRNA export.
Utp8p Binds tRNA Directly and Saturably in Vitro—Substrate-induced intrinsic fluorescence quenching of Tyr and Trp residues was used to investigate whether Utp8p can interact with tRNA. The analysis showed that Utp8p binds mature tRNA directly and saturably with a calculated Kd of 11 µM (Fig. 8). This finding indicated that Utp8p has a tRNA-binding site. Utp8p also interacts with the E. coli 5 S rRNA but to a lower extent compared with that observed for tRNA binding. Furthermore, saturable binding to 5 S rRNA could not be achieved under the conditions employed, suggesting that the protein is interacting non-specifically with the 5 S rRNA. This property of Utp8p is not unusual, because it is well established that bona fide eukaryotic and prokaryotic tRNA-binding proteins interact non-specifically with non-cognate RNAs in vitro (43–46). Taken together the data suggest that Utp8p has an RNA-binding site that is specific for tRNA.
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Utp8p Does Not Appear to Function as a Nuclear tRNA Export Receptor—To ascertain whether Utp8p is a nuclear tRNA export receptor, a heterokaryon shuttling assay was used to investigate whether Utp8p shuttles between the nucleus and cytoplasm (19, 26). Xpo1p, a nuclear receptor that is involved in nuclear export of proteins containing a leucine-rich nuclear export signal, was used as a control for a protein known to shuttle between the nucleus and cytoplasm (25). The heterokaryon assay involves monitoring the movement of a protein from a donor nucleus to a recipient nucleus in heterokaryons. To avoid nuclear import of cytoplasmic Utp8p and Xpo1p into the recipient nucleus, the donor strain harboring a low copy number plasmid with UTP8-GFP or XPO1-GFP under the control of the GAL1 promoter was first grown in medium containing galactose to induce expression of the fusion proteins, and then briefly in medium containing glucose to repress the GAL1 promoter. The donor strain was then mated with a kar1-1 mutant strain, which is defective in nuclear fusion. Movement of Utp8-GFP and Xpo1-GFP between nuclei was monitored by direct fluorescence microscopy (Fig. 9). Utp8-GFP was found in a single nucleus in essentially all heterokaryons analyzed over a 5-h period after mating was initiated. In contrast, Xpo1-GFP was present in both nuclei of heterokaryons over the same period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To identify components of the S. cerevisiae nuclear tRNA export machinery, we used a yeast tRNA three-hybrid selection method to screen a cDNA library for genes of proteins that interact with tRNA, and an in vivo nuclear tRNA export assay based on amber suppression to ascertain whether the identified proteins are playing a role in nuclear tRNA export. This strategy resulted in the identification of Utp8p, an essential 80-kDa nucleolar protein. Utp8p plays a role in nuclear export of both aminoacylated and non-aminoacylated tRNAs, and it appears to act at a step between tRNA maturation/aminoacylation and tRNA translocation out of the nucleus.
Utp8p was recently identified as part of a protein complex associated with the U3 small nucleolar RNA, which is involved in processing of pre-18 S rRNA (22). Depletion of Utp8p for an extended period was reported to cause a reduction in the amount of 18 S rRNA but not 25 S rRNA. This finding led to the suggestion that Utp8p may play a role in 18 S rRNA biogenesis. Western blot analyses indicated that Utp8p is maximally depleted within 6 h (Fig. 3B); despite carrying out the depletion for 24 h, we could not detect any significant changes in the level of 18 S rRNA (Fig. 4C). Furthermore, no defect was observed for nuclear export of rpL3 and rpL25, two ribosomal subunits that associate with the 35 S pre-rRNA during the early stages of ribosomal biogenesis in the nucleus (Fig. 4B). However, a block in nuclear tRNA export was observed after depletion of Utp8p for 6 h (Fig. 3C). Although these results do not exclude the involvement of Utp8p is pre-18 S rRNA maturation, they strongly suggest that the major function of Utp8p is in nuclear tRNA export and not 18 S rRNA biogenesis. The significance of the involvement of Utp8p in both 18 S rRNA synthesis and nuclear tRNA export is not understood and will require further studies.
Similar to Los1p and exportin-t, overexpression of Utp8p restored export of
mutants
defective in nuclear export (Fig. 2,
A and B)
(21). Furthermore, loss of
Utp8p function blocked nuclear export of mature tRNAs derived from both
intronless and intron-containing pre-tRNAs
(Fig. 3C) but not tRNA
aminoacylation in the nucleus (Fig.
6). Utp8p provided in trans also alleviated nuclear
retention of tRNATyr in the tys1ts mutant
strain expressing a catalytically active TyrRS enzyme that is defective in
nuclear import (Fig. 7). These
results suggest that Utp8p could be functioning as a nuclear tRNA export
receptor for both the aminoacylation-dependent and -independent nuclear tRNA
export pathways operating in S. cerevisiae. A characteristic of
nuclear import/export receptors is that they shuttle between the nucleus and
cytoplasm. Thus, a heterokaryon shuttling assay was used to ascertain whether
Utp8p shuttles between the nucleus and cytoplasm. This assay is well
documented and has been used to show that Cca1p plays a role in nuclear tRNA
export in S. cerevisiae
(19,
26). Unlike the nuclear export
receptor Xpo1p, Utp8p was found in only one nucleus of heterokaryons
(Fig. 9). This suggests that
either Utp8p does not shuttle between the nucleus and cytoplasm or it shuttles
so slowly that the amount of the protein in the recipient nucleus could not be
detected. However, the most likely explanation based on the finding that
neither Los1p nor exportin-t could compensate for loss of Utp8p function (data
not shown) is that Utp8p is not functioning as a nuclear tRNA export receptor.
These results combined with the finding that depletion of Utp8p did not affect
tRNA processing/modification (Fig.
5) and aminoacylation of tRNAs derived from intronless and
intron-containing pre-tRNAs in the nucleus
(Fig. 6) suggest that Utp8p
acts at a step between tRNA maturation/aminoacylation and translocation of the
tRNA to the cytoplasm. This step appears to be located in the nucleolus,
because the protein is found in this compartment at steady state (data not
shown (22)).
Maturation of pre-tRNAs is a multistep process that occurs in the nucleolus, nucleoplasm, and nuclear envelope in an order that is undefined (35, 49–51). Recently, FISH analysis detected mature tRNAs derived from intronless and intron-containing pre-tRNAs in the nucleolus of S. cerevisiae defective in nuclear tRNA export because of a block in nuclear tRNA aminoacylation or loss of Los1p function (17). This finding suggests that tRNAs are taken to the nucleolus for final maturation and/or aminoacylation before they are exported to the cytoplasm. This observation also implies that the nucleolus is the starting point for the tRNA export process. It is possible that Utp8p is responsible for initiating tRNA export by enabling the tRNA to gain access to the components involved in translocation of the tRNA across the NPC.
Channeling is a mechanism used to spatially compartmentalize biochemical processes. This is achieved by directly transferring a substrate from one component to another within a multistep biochemical pathway. A channeling mechanism is used in mRNA and ribosome biogenesis and export, tRNA maturation, delivery of cytoplasmic tRNAs to aminoacyl-tRNA synthetases, and transfer of aminoacyl-tRNAs from aminoacyl-tRNA synthetases to ribosomes (4, 35, 48, 49, 52, 53, 54–57). This type of mechanism may be used to link the tRNA maturation system to the nuclear export apparatus. Because Utp8p binds tRNA directly (Fig. 8), we suggest that the protein may link tRNA maturation and export by functioning as an intranuclear factor that picks up both aminoacylated and non-aminoacylated tRNAs from the nucleolus and delivers them directly to the export receptors of the aminoacylation-dependent and -independent pathways, or indirectly by delivering the tRNAs to the next component(s) of the export pathways. To address this possibility, a yeast two-hybrid assay was used to test whether Utp8p interacts with Los1p or TyrRS. However, no interaction between Utp8p, Los1p, or TyrRS was observed (data not shown). This finding does not negate the proposed role of Utp8p, because it is possible that the interaction between Utp8p and Los1p or TyrRS is transient or too weak to be detected by the yeast two-hybrid interaction assay.
Recent studies have shown that Cca1p is directly involved in nuclear tRNA export in S. cerevisiae (19). Like methionyl-tRNA synthetase, overexpression of Cca1p restored nuclear export of tRNAMet in a los1 strain. In addition, Cca1p was shown to shuttle between the nucleus and cytoplasm. The fact that tRNAMet is derived from intronless pre-tRNA led to the suggestion that Cca1p is required for nuclear export of non-aminoacylated tRNAs derived from intronless pre-tRNAs (19). We have shown that overexpression of wild type TyrRS or Utp8p but not Cca1p restored nuclear export of tRNATyr, a tRNA made from intron-containing pre-tRNA, in the tys1ts strain expressing the nuclear import-defective TyrRS mutant protein (Fig. 7). This observation supports the conclusion that Cca1p is involved in nuclear export of tRNAs from intronless pre-tRNAs and suggests that export of non-aminoacylated tRNAs derived from intron-containing tRNAs is facilitated by an unidentified protein.
Finally, we have established a biochemical method to assess directly the aminoacylation status of tRNAs in the nucleus. This approach is an adaptation of published methods and allows for the preparation of aminoacylated tRNAs from isolated nuclei and separation of the aminoacylated and non-aminoacylated forms of a tRNA by PAGE (21, 31, 32, 34). The method described can be used to ascertain whether all tRNAs or certain tRNAs are aminoacylated in the nucleus. This information will be necessary to establish whether both the aminoacylation-dependent and -independent export pathways are used concurrently in S. cerevisiae. Furthermore, it can be used routinely to ascertain whether a block in nuclear tRNA export resulting from chromosomal mutations is due to a defect in nuclear tRNA aminoacylation. Because this electrophoretic method can distinguish between modified and unmodified tRNA, it will also be useful for analysis of the effect of chromosomal mutations on tRNA modification (Fig. 5B).
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Tel.: 519-824-4120 (ext. 53432); Fax: 519-766-1499; E-mail: mangroo{at}chembio.uoguelph.ca.
1 The abbreviations used are: NPC, nuclear pore complex; TyrRS, tyrosyl-tRNA
synthetase; FISH, fluorescence in situ hybridization; ORF, open
reading frame; GFP, green fluorescent protein; snoRNA, small nucleolar
RNA.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) was performed as described under
"Experimental Procedures." An Eadie-Hofstee plot was used to
calculate the tRNA-binding affinity of Utp8p.