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INTRODUCTION |
Upstream open reading frames
(uORFs)1 in the 5'-leader
regions of eukaryotic and prokaryotic transcripts can serve critical regulatory functions (1-4). The fungal mRNAs specifying the small subunit of carbamoyl phosphate synthetase contain a uORF encoding an
evolutionarily conserved peptide (Fig. 1A). In the cases of Saccharomyces cerevisiae CPA1 and Neurospora crassa
arg-2, the capacity to translate this uORF peptide is essential
for establishing Arg-specific negative regulation of gene expression
in vivo (5-9). Evolutionarily conserved uORFs are also
found in mammalian mRNAs including those specifying HER-2/NEU (see
Ref. 10, and references therein), BCL-2 (11, 12), and
CCAAT/enhancer-binding protein (13). While these mammalian uORFs affect
translation, their regulatory roles remain unknown. Understanding how
the CPA1 and arg-2 uORFs exert regulatory effects
should provide insight into such uORF-mediated control mechanisms.
The evidence for a role of CPA1 uORF translation in
regulation is based on mutational studies (see Ref. 14, and references therein) and a variety of regulatory models are consistent with the
existing data concerning the CPA1 uORF (14). For
arg-2, mutational studies have been combined with direct
biochemical studies. Addition of Arg to growing cells causes a rapid
decrease in the rate of ARG-2 polypeptide synthesis and a decrease in
the association of the arg-2 mRNA with ribosomes (7).
Experiments with reporter genes show that translation of the wild-type
arg-2 uORF is critical for Arg-specific translational
control. mRNAs in which uORF translation is eliminated or in which
the evolutionarily conserved peptide sequence is altered no longer show
decreased association with ribosomes when Arg is added to cells (8,
9).
Further insight into the mechanism of Arg-specific translational
attenuation mediated by the arg-2 uORF-encoded peptide was gained using cap-, poly(A)-, and amino acid-dependent
translation extracts derived from N. crassa in which
regulation is reconstituted (15). Using reaction mixtures containing
low or high Arg concentrations and a primer extension inhibition
(toeprint) assay to map the positions of ribosomes on capped and
polyadenylated synthetic RNA templates, high Arg concentrations are
observed to cause ribosome stalling with the wild-type uORF termination
codon at the ribosomal A site (16). Since reinitiation following uORF
translation does not appear to be efficient in this case, these data
provide the basis for a regulatory model in which the Arg-stalled
ribosomes prevent trailing scanning ribosomes from reaching the
downstream start codon (16, 17).
Based on its cis-acting ability to repress the translation
of downstream RNA sequences, the arg-2 uORF-encoded peptide
was named the Arg attenuator peptide (AAP). Dissection of sequences outside of the 24-amino acid coding region established that little else
of the original arg-2 mRNA is required for the AAP's
regulatory function (18). Neither deletion of the intercistronic
sequences nor changing the UAA termination codon to UGA or UAG alters
the AAP's regulatory capacity. Furthermore, direct fusion of the AAP at the N terminus of a polypeptide results in Arg-specific stalling of
ribosomes involved in elongation in the region immediately downstream
of the AAP, indicating that the termination codon is dispensable for stalling.
Here we examined the generality of AAP-mediated regulation by studying
the function of the CPA1 and arg-2 AAPs in
translation extracts derived from S. cerevisiae, N. crassa, and wheat germ. Both CPA1 and arg-2
AAPs mediated translational attenuation in each of these systems as
determined by LUC assays. Thus, factors that permit AAP-mediated
translational attenuation can be found in plant as well as fungal
systems. In the S. cerevisiae and N. crassa
translation extracts, primer extension inhibition (toeprint) assays
indicated that Arg-specific translational attenuation was associated
with the stalling of ribosomes after AAP translation. The level of
charged Arg-tRNA did not appear to be responsible for effecting
Arg-specific control because the tRNA was fully charged even at low Arg
concentrations. In contrast, all other well understood examples of
translational regulation of amino acid biosynthetic genes in eukaryotes
and prokaryotes that are mediated by uORFs respond to the level of tRNA
charging (19, 20). Thus, AAP-mediated ribosome stalling appears to be
an evolutionarily conserved cis-acting control mechanism
that regulates the expression of a fungal Arg-biosynthetic gene in
response to Arg independent of the level of charged tRNA.
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EXPERIMENTAL PROCEDURES |
Templates for RNA Synthesis--
Linearized plasmid templates
were designed to produce capped and polyadenylated synthetic RNAs
encoding firefly LUC. The first type of RNA was designed to contain the
entire S. cerevisiae CPA1 uORF and intercistronic region in
its 5'-leader (pAG101; Fig. 1B, Table I). The second type of
RNA was designed to have the CPA1 uORF coding sequence fused
directly in-frame with the LUC open reading frame (pAG102; Fig.
1C, Table I). Also constructed were mutant variants of each
type containing either an Asp to Asn codon change at codon 13 of the
CPA1 uORF (pAG103 and pAG104) or a Met to Leu codon change
(AUG 
UUG) at the predicted CPA1 uORF translation
initiation codon (pAG105) by using PCR-based procedures (9). PCR
products were placed into the pHLUC+NFS4 vector (15). Primers for PCR
reactions were: AG1 (5'-TGTTGAAGATCTACCCTTTTTGCAGATTTG-3'), which includes a 5'-BglII site, used for pAG101, pAG102,
pAG103, and pAG104; AG3 (5'-ATCTGACCATGGTTGAAATATTTTTAGGAGTGGTT-3'),
which includes a 3'-NcoI site, used for pAG101, pAG103, and
pAG105; AG4 (5'-ATAGATGGTGACCTGGTGGGAGCTAGTTTTCCA-3'), which includes a
3'-BstEII site, used for pAG102, pAG104, and pAG106; AG5
(5'-CAGATATGTAGTTTTGGCAGG-3'), which contains the Asp to Asn codon
change at codon 13 of the CPA1 uORF, used for pAG103 and
pAG104; and AG6
(5'-TGTTGAAGATCTACCCTTTTTGCAGATTTGAAATAAAAAAAACATTATTTGTTTAGCTTAT-3'), which contains a 5'-BglII site and changes the
predicted uORF translation initiation codon, used for pAG105 and
pAG106. Corresponding templates for the synthesis of RNA containing the
N. crassa arg-2 AAP in the 5'-leader region (Table I) were
described previously (15, 16, 18), as was the template used to produce
capped and adenylated synthetic mRNA encoding sea pansy LUC to
serve as an internal control for translation reactions (18).
Plasmid DNA templates were purified by equilibrium centrifugation or by
using a plasmid purification kit from Qiagen; capped, polyadenylated
RNA was synthesized with T7 RNA polymerase from EcoRI-linearized plasmid DNA templates, and the yield of RNA
was quantified (15).
Cell-free Translation and Primer Extension Inhibition (Toeprint)
Analyses--
The preparation of translation extracts was as described
(21) from S. cerevisiae strain YAS1874
(MATa MAK10::URA3 PEP4::HIS3
prb1 prc1 ade2 leu2 trp1 his3 ura3) (22) with two modifications:
buffer A was pH 7.6 instead of pH 7.4, and extracts were treated with
micrococcal nuclease immediately after recovery from the Sephadex G-25
column, prior to freezing and storage. Nuclease-treated yeast extracts
were used because nuclease treatment did not significantly affect amino
acid-dependence or Arg-specific regulation under our assay conditions,
but in initial comparative studies greatly increased the absolute level
of reporter RNA translation and yielded superior toeprints (data not
shown). The preparation of translation extracts from N. crassa (with no nuclease treatment) was as described (15).
The reaction conditions for in vitro translation using
S. cerevisiae and N. crassa extracts were
essentially as described previously (15, 21). For translation in
S. cerevisiae extracts, the final concentrations of
K+ and Mg2+ were 230 and 3.4 mM,
respectively. Translation reaction conditions using nuclease-treated
wheat germ extracts (Promega) were essentially those specified by the
supplier, except that, to achieve maximum activity, K+ and
Mg2+ final concentrations were adjusted to 100 and 2.1 mM, respectively. All reaction mixtures were incubated at
25 °C; for LUC assays, translation was halted by freezing in liquid
nitrogen after 30 min of incubation, and 5-µl aliquots of the
ice-thawed mixtures were used for analysis (15, 18).
A wide range of conditions were examined to find those that were
optimal for toeprinting in S. cerevisiae-derived reactions, including pH, heat pretreatment, reaction temperature, and
Mg2+ concentration (23). These were similar to those
earlier determined to be optimal for N. crassa-derived
reactions (data not shown). Therefore, the toeprint assays of both
S. cerevisiae- and N. crassa-derived reaction
mixtures were accomplished after incubation as described in the text
using primer ZW4 and the previously established method (16). All
toeprint data shown are representative of multiple experiments.
Measurement of tRNA Aminoacylation--
The assays for tRNA
aminoacylation were adapted from a previously described procedure (24).
Translation reaction mixtures with total volumes of 100 µl (S. cerevisiae and N. crassa) or 60 µl (wheat germ)
containing different concentrations of Arg were incubated for 10 min.
Then aliquots of 90 µl (S. cerevisiae and N. crassa) or 54 µl (wheat germ) were removed (the remainder of
translation reaction mixtures were incubated to the 30-min time point,
then used for LUC assays) and immediately added to ice-cold tubes
containing a mixture of 300 µl of phenol (pH 4.5) and 200 µl of
sodium acetate (pH 4.5). Tubes were vortexed for 60 s and then
centrifuged for 20 min. The aqueous layers were transferred to new
tubes and mixed with 2.5 volumes of ethanol. Tubes were frozen at
80 °C for at least 15 min, and then the total nucleic acids were
recovered by centrifugation for 20 min. The nucleic acid pellet was
dissolved in 20 µl of 10 mM sodium acetate, 1 mM EDTA (pH 4.5). An aliquot (2.0 µl) was used to measure A260; immediately prior to gel electrophoresis,
nucleic acids were adjusted to a final concentration of 2.5 µg/µl
(assuming 40 µg of nucleic acid/A260) by the
addition of acid gel loading buffer (24).
The level of tRNA charging initially present in extracts at time
T0 was determined by processing as described above of 50 µl of extract (S. cerevisiae and N. crassa) or
30 µl of extract (wheat germ) with no addition of other reaction
mixture components (e.g. additional salt, synthetic
mRNA, and amino acids in all three cases, plus an energy-
regenerating system for S. cerevisiae and N. crassa). As an additional control, tRNAs in T0
extracts were deacylated by alkali treatment. First, aliquots of
T0 extracts (50 or 30 µl) were mixed with 250 µl of
0.2 M Tris-HCl (pH 8.0) and extracted with 300 µl of
phenol (pH 8.0). Nucleic acids in the aqueous phase were precipitated
with salt and ethanol; the precipitates were dissolved in 100 µl of
0.1 M Tris-HCl (pH 8.8) and incubated at 37 °C for 20 min to deacylate the tRNAs. After another ethanol precipitation, the
pellet was dissolved in 20 µl of 10 mM sodium acetate, 1 mM EDTA (pH 4.5) prior to dilution with acid gel loading buffer.
The procedures for acid/urea gel electrophoresis, electrophoretic
transfer, and Northern blot hybridization to identify charged and
uncharged tRNAs were essentially as described (24), except that
denatured salmon sperm DNA was not included in the prehybridization and
hybridization solutions, and the membranes after hybridization were
exposed to screens of a Molecular Dynamics PhosphorImager for
approximately 4 h. DNA probes are the reverse complements of these
tRNA regions: JA11: 5'-TCGGTTTCGATCCGAGGACATCAGGGTTATGA-3', complement to 32-63 of S. cerevisiae
tRNAiMet (25,
26)2; ZW32:
5'-ACGATGGGGGTCGAACCC-3', complement to 50-67 of S. cerevisiae Arg-tRNA 3a and 3b (27); ZW33:
5'-TGGTTCGCAGCCAGACGC-3', complement to 24-41 of S. cerevisiae Arg-tRNA 2 (28); ZW34: 5'-ATCTTCTGGTTCGCAGCC-3', complement to 30-47 of S. cerevisiae Arg-tRNA 2; ZW38:
5'-ACCACGCTGGGAGTCGAACC-3', complement to 52-71 of wheat germ tRNA-Arg
(CCG) (29); ZW39: 5'-ACTCCGCTGGGGATCGAACC-3', complement to 52-71 of
wheat germ tRNA-Arg (ICG); (30); ZW40: 5'-TGGGACCTGTGGGTTATGGG-3',
complement to 31-50 of wheat germ
tRNAiMet (31); ZW41:
5'-TCGATCCTGGGACCTGTGG-3', complement to 39-57 of wheat germ
tRNAiMet; ZW42:
5'-ACCTCCGGGTTATGAGCCC-3', complement to 28-46 of N. crassa tRNAiMet (32); ZW43:
5'-TCGAGTGACCTCCGGGTT-3', complement to 36-53 of N. crassa
tRNAiMet; ZW44:
5'-CTTCAGTCTGACGCTCTCCC-3', complement to 18-37 of N. crassa tRNA-Phe (GAA) (33); ZW45: 5'-TGCGGTTTGTGTGGATCG-3', complement to 56-73 of N. crassa tRNA-Phe (GAA).
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RESULTS |
AAP-mediated Arg-specific Translational Attenuation in Three
Cell-free Translation Systems--
We examined Arg-specific regulation
mediated by the S. cerevisiae and N. crassa AAPs
in translation extracts derived from S. cerevisiae, N. crassa and wheat germ. Capped and polyadenylated synthetic RNAs
were synthesized from templates in which the S. cerevisiae
CPA1 AAP or the N. crassa arg-2 AAP were placed
upstream of firefly LUC, either as uORFs or as in-frame N-terminal
extensions (Fig. 1, B and
C, Table I). Equal amounts of
each RNA were used to program translation extracts. As an internal
control, a second capped and polyadenylated synthetic RNA that encoded
sea pansy LUC (which lacked fungal regulatory sequences) was also added to the extracts (18). For each of the extracts used, the addition of 10 µM each of the 20 amino acids to reaction mixtures was
sufficient for near maximal translation of LUC (data not shown).
Additional Arg could be added to translation extracts with relatively
slight effects on protein synthesis from RNA templates lacking
arg-2 regulatory sequences (e.g. sea pansy LUC,
Fig. 5B).
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Fig. 1.
Sequences of the fungal AAPs and the 5'
leader regions of CPA1-LUC genes used in this study
(also see Table I). A, comparisons of the AAPs from
S. cerevisiae (Sc; Ref. 5), N. crassa
(Nc; Ref. 52), Magnaporthe grisea (Mg;
Ref. 61), Trichoderma virens (Tv; Ref. 62), and
Aspergillus nidulans (An; GenBank accession no.
AJ224085). B, sequences of wild-type and mutant templates in
which the CPA1 AAP is encoded by a uORF. The sequence shown
begins with the T7 RNA polymerase-binding site and ends within the LUC
coding region (16). The 5' and 3' boundaries of the CPA1
region that was amplified by PCR are boxed. The amino acid
sequences of the CPA1 AAP and the N terminus of LUC are
indicated. Point mutations are shown below the wild-type sequence. The
sequence for which the reverse complement was synthesized and used as
primer ZW4 for toeprint analysis is indicated by a
horizontal arrow below the sequence.
C, sequences of wild-type and mutant templates containing
CPA1 AAP-LUC fusion genes. The sequence shown begins with
the T7 RNA polymerase-binding site and ends within the LUC coding
region (57). The 5' and 3' boundaries of the CPA1 region
that was amplified by PCR are boxed. The amino acid sequence
of the N terminus of the AAPsc-LUC fusion polypeptide is
indicated. Point mutations are shown below the wild-type sequence. The
sequence for which the reverse complement was synthesized and used as
primer ZW4 for toeprint analysis is indicated by a
horizontal arrow below the sequence.
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Comparisons of the translation of firefly LUC in reaction mixtures
supplemented with low (10 µM) or high (2 mM)
Arg showed that the wild-type CPA1 AAP, when present as a
uORF (Fig. 1B), reduced the translation of LUC when the
concentration of Arg was high (Table I). Translation of LUC from RNA
containing the wild-type N. crassa arg-2 AAP was also
subject to Arg-specific negative regulation in each extract (Table I).
Introduction of the D13N mutation in the S. cerevisiae AAP
coding region (Fig. 1B) or the corresponding D12N mutation
into the N. crassa coding region eliminated this regulatory
effect in all cases (Table I).
The wild-type CPA1 and arg-2 AAPs, when fused
directly to LUC as N-terminal extensions (Fig. 1C), also
functioned to regulate translation in all three extracts (Table I). The
LUC polypeptide produced appeared to initiate at the AAP start codon
because changing this codon from AUG to UUG resulted in unregulated and
substantially reduced (50-fold in N. crassa, 30-fold in
S. cerevisiae) LUC synthesis (data not shown). The
CPA1 AAP D13N mutation and the arg-2 AAP D12N
mutation eliminated the regulatory effect of Arg in all three systems,
showing the strong dependence of Arg regulation on the sequence of the
AAP peptide and the lack of necessity of a uORF termination codon for regulation.
Ribosomal Stalling in High Arg Is Mediated by the Wild-type CPA1
and arg-2 AAPs--
Toeprint assays, in which reverse transcriptase is
used for primer extension in translation extracts, enables the mapping of the positions of N. crassa ribosomes on RNA (16, 18).
Here we applied this technique to S. cerevisiae ribosomes.
To our knowledge, this is the first use of toeprinting to examine the
positions of S. cerevisiae ribosomes on RNA. Primer
extension from RNA templates containing the CPA1 uORF in the
absence of extract yielded cDNA products predominantly
corresponding to full-length extension of the primer as well as other
shorter transcription products (Fig. 2,
lanes 8 and 15). The shorter products
are produced in relatively much lower quantities when the RNA is
reverse-transcribed in buffer formulated for reverse transcription
(data not shown) rather than the buffer formulated for in
vitro translation necessary for these experiments. Extracts
that were not programmed with RNA did not yield any of these signals
(Fig. 2, lanes 7 and 16), as predicted
if these represented the products obtained from priming on the
synthetic RNA template. When RNA containing the CPA1 uORF in
its 5'-leader was used to program S. cerevisiae extracts
containing high Arg (500 or 2000 µM, Fig. 2,
lanes 2 and 3) but not low Arg (10 µM, Fig. 2, lane 1), new signals
were observed that corresponded to ribosomes stalled with the uORF
termination codon in the ribosome A site (confirmed by high resolution
mapping on other gels, data not shown). The effect of Arg to stall
ribosomes at the termination codon increased when the concentration of
Arg was raised from 500 µM to 2000 µM. Arg
also caused ribosome stalling at the CPA1 uORF termination
codon in N. crassa extracts (Fig. 2, compare lanes 10 and 11 to lane
9). The CPA1 AAP D13N mutation, which eliminates
regulation in vivo, (5), eliminated Arg-specific effects on
toeprints in both extracts (Fig. 2, lanes 4-6
and 12-14), consistent with the loss of regulation observed
by LUC assay (Table I).
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Fig. 2.
Effects of the CPA1 AAP
encoded as a uORF on Arg-specific regulation in translation extracts
derived from S. cerevisiae and N. crassa. Equal amounts (120 ng) of synthetic RNA
transcripts were used to program translation mixtures derived from
S. cerevisiae or N. crassa. The transcripts
encoded either the wild-type (wt) or D13N mutant
CPA1 AAP as a uORF in the 5' leader as indicated. The
20-µl reaction mixtures were supplemented with different
concentrations of Arg (10, 500, or 2000 µM as indicated)
and with 10 µM each of the other 19 amino acids. After 20 min of incubation at 25 °C, the reaction mixtures were toeprinted
with primer ZW4 as described (16). The products obtained from primer
extension of pure RNA (18 ng) in the absence of translation reaction
mixture (-EXT) and from a translation reaction mixture not
programmed with RNA (-RNA) are shown for comparison. The
arrows indicate the positions of premature transcription
termination products corresponding to ribosomes bound at
AUGuORF, UAAuORF, or AUGLUC. The
arrowhead indicates the position of an additional toeprint
site upstream of UAAuORF observed in N. crassa
extracts containing high Arg concentrations; asterisks
indicate an additional toeprint site downstream of UAAuORF
observed in S. cerevisiae and N. crassa extracts
containing high Arg concentrations. The bracket indicates
additional toeprints observed downstream of UAAuORF in
S. cerevisiae extracts containing high Arg concentrations.
The star indicates a strong signal observed from primer
extension of RNA in the absence of extract (-EXT).
Dideoxynucleotide sequencing reactions for the wild-type
CPA1 template are shown on the left; the
nucleotide complementary to the dideoxynucleotide added to each
sequencing reaction is indicated below the corresponding
lane so that the sequence of the template can be directly deduced; the
5'-to-3' sequence reads from top to bottom.
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Puromycin, an inhibitor of translation that releases 80 S ribosomes
from RNA, would be expected to release Arg-specific signals if they
arose from the stalling of ribosomes. Therefore, extracts were
programmed with RNA and incubated for 15 min in low or high Arg; then
puromycin was added to a final concentration of 1.3 mM (or
water was added as a negative control) and incubation continued for 5 min. Extracts were then subjected to toeprint analysis. Puromycin
released the Arg-specific toeprints observed in both S. cerevisiae and N. crassa extracts (data not shown).
Thus, the Arg-specific signals appear to be a reversible consequence of the association of ribosomes with the RNA.
In N. crassa extracts, signals corresponding to ribosomes
with the uORF and LUC initiation codons in their P-sites were observed in extracts programmed with RNA. For constructs containing the wild-type but not the D13N uORF, these signals were reduced when Arg
was added. Similar results are observed for the wild-type arg-2 uORF in N. crassa extracts and are
interpreted to arise as a consequence of ribosome stalling at the uORF
termination codon, which decreases ribosome loading at the LUC AUG, and
decreases the capacity to detect signal at the uORF AUG (16, 18).
The wild-type CPA1 AAP caused additional Arg-specific
effects in each extract, some common and some system-specific (Fig. 2
and data not shown). In N. crassa extracts, an additional
signal (arrowhead) approximately 30 nucleotides upstream of
the stop codon appeared in high Arg. These possibly represent ribosomes queued behind ribosomes that have stalled at the uORF termination codon
(16, 18). In both systems, Arg caused a substantial increase in
(puromycin-releasable) toeprints in the intercistronic region
(asterisk, approximately 12 and 16 nucleotides downstream of
the stop codon in N. crassa, and 16 nucleotides downstream of the stop codon in S. cerevisiae). In S. cerevisiae, additional Arg-regulated toeprints were observed
further downstream in the intercistronic region (star and
bracket). One of these signals corresponds in position to a
strong signal in the intercistronic region of the RNA, which was also
present in primer extension products obtained from the RNA in the
absence of translation extract (Fig. 2, lanes 8 and 15, star). The physical basis for these
additional bands, which may arise for reasons similar or different than
those responsible for the "echo band" phenomenon, in which a
ribosome located at an initiation codon can cause a primary toeprint
and an additional toeprint (23), remain to be elucidated. Possibly, they could represent ribosomes or additional machinery recruited by
ribosomes; alternatively, they could reflect increased secondary structure in the RNA arising as a consequence of ribosome binding.
In a manner highly similar to the wild-type CPA1 AAP, the
wild-type arg-2 AAP (in these experiments placed in an
improved initiation context; Ref. 16) caused ribosomes to stall at the uORF termination codon in response to Arg in both S. cerevisiae and N. crassa systems; the D12N mutation
eliminated regulation (Fig. 3). Thus, the
two fungal AAPs acted similarly when present as uORFs to stall
ribosomes in S. cerevisiae and N. crassa
systems.
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Fig. 3.
Effects of the arg-2 AAP
encoded as a uORF on Arg-specific regulation in translation extracts
derived from N. crassa and S. cerevisiae. Equal amounts of synthetic RNA transcripts
(120 ng) were translated in reaction mixtures and analyzed by
toeprinting as described in the legend to Fig. 2. The transcripts
encoded either the wild-type (wt) AAP in an improved
initiation context or the D12N AAP (16). Arrows indicate the
positions of premature transcription termination products
corresponding to ribosomes bound at
AUGuORF, UAAuORF, or
AUGLUC. The arrowhead indicates the position of
an additional toeprint site upstream of UAAuORF observed in
N. crassa extracts containing high Arg concentrations. The
star indicates a signal observed from primer extension of
RNA in the absence of extract (-EXT) that coincides with a
toeprint signal that increases with high Arg (16).
Dideoxynucleotide sequencing reactions for the wild-type
arg-2 template are shown on the left; the
nucleotide complementary to the dideoxynucleotide added to each
sequencing reaction is indicated below the corresponding
lane so that the sequence of the template can be directly deduced; the
5'-to-3' sequence reads from top to bottom.
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While AAP-dependent regulation was observed using wheat
germ extracts (Table I), in primer extension experiments, no signals indicating Arg-specific stalling were apparent (data not shown). All
signals, including full-length cDNA products, were weaker in primer
extension analyses using wheat germ. This failure to achieve results in
the wheat germ system that were comparable to those obtained with the
fungal systems is possibly attributable to the presence of an RNase H
activity in wheat germ extracts (34).
The effect of Arg-specific, AAP-mediated regulation on ribosomes
involved in elongation was tested using CPA1 AAP-LUC and arg-2 AAP-LUC fusions in S. cerevisiae and
N. crassa extracts. A high concentration of Arg
substantially increased the intensity of a series of toeprints on the
CPA1 AAP-LUC RNA in both extracts (Fig.
4, compare lanes 4 and 3, and lanes 12 and
11). The D13N mutation eliminated these Arg-specific effects
on toeprints (Fig. 4, compare lanes 1 and
2, and lanes 9 and 10), as
did treatment with puromycin after 15 min, as described above (Fig. 4,
lanes 5, 6, 13, and
14), indicating that they resulted from an interaction of
ribosomes with the RNA. In both extracts, the most 5'-proximal of the
Arg-specific toeprints corresponded to ribosomes translating the first
codon following the AAP coding sequence (Fig. 4,
arrowheads). This stall site in the fusion polypeptide
corresponds to the position, relative to the CPA1 AAP coding
sequence, of the uORF termination codon. This toeprint site was
followed by additional Arg-induced toeprints corresponding to ribosomes
stalled in the downstream LUC coding region (indicated by
brackets). These signals extend further downstream in
S. cerevisiae extracts.
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Fig. 4.
Effects of the CPA1 AAP as
an N-terminal fusion to LUC on Arg-specific regulation in translation
extracts derived from S. cerevisiae and N. crassa. Equal amounts of synthetic RNA transcripts (120 ng) were translated in reaction mixtures and analyzed by toeprinting as
described in Fig. 2. The transcripts encoded either the wild-type
(wt) AAP-LUC fusion or the D13N mutant AAP-LUC fusion as
indicated. Puromycin (Pur) was added where indicated (+), as
described in the text. The arrow indicates the position of
premature transcription termination products corresponding to ribosomes
bound at the AAP initiation codon (AUGAAP). The
arrowhead indicates the positions of premature termination
products corresponding to ribosomes stalled at the codon immediately
following the last codon of the AAP in S. cerevisiae and
N. crassa extracts containing a high Arg concentration. The
bracket indicates the position of premature termination
products corresponding to ribosomes stalled in the LUC coding region in
S. cerevisiae and N. crassa extracts containing a
high Arg concentration. Dideoxynucleotide sequencing reactions for the
wild-type CPA1 AAP-LUC fusion template are shown on the
left; the nucleotide complementary to the dideoxynucleotide
added to each sequencing reaction is indicated below the
corresponding lane so that the sequence of the template can be directly
deduced; the 5'-to-3' sequence reads from top to bottom.
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The length of the region in which ribosomes involved in elongation were
stalled in response to Arg appeared to be determined by the source of
the extract and not the source of the AAP. Both CPA1 and
arg-2 AAPs yielded a more extended series of toeprint sites
in S. cerevisiae than N. crassa (Fig. 4; data not
shown). The reasons for these extract-dependent differences
in toeprinting are not known but might reflect faster translation
elongation rates in S. cerevisiae-derived extracts (data not shown).
Arg-specific Regulation Appears Independent of the Charging Status
of Arginyl-tRNAs--
When the effect of Arg on ribosome stalling on
transcripts encoding the wild-type arg-2 AAP-LUC fusion was
examined in N. crassa translation extracts, the amount of
stalling increased as the concentration of Arg increased (Fig.
5A, lanes
1-5). Similar effects were observed with the
CPA1 AAP-Luc fusion in N. crassa extracts and
with both fusions in S. cerevisiae extracts (data not
shown). The S. cerevisiae D13N and N. crassa D12N
mutants did not show stalling at any Arg concentration in either
extract (Fig. 4; Fig. 5A, lanes 6-8;
data not shown). Consistent with the observed increase in stalling of
ribosomes on RNA containing the wild-type AAP-LUC fusion, the magnitude
of Arg-specific regulation increased in S. cerevisiae,
N. crassa, and wheat germ extracts as the concentration of
added Arg was increased from 10 µM to 5 mM
(Fig. 5B) as determined by LUC assay. It should be noted that, while the precision of measurements in a given experiment is
high, the absolute magnitude of regulation by Arg differs between extract preparations and experiments (e.g. the extracts used
in the experiment shown in Table I showed a lower magnitude effect than
those used in Fig. 5B). Nonetheless, in multiple experiments using any of the amino acid-dependent fungal extracts
prepared in our laboratory to date (28 independently prepared N. crassa extracts and 12 independently prepared S. cerevisiae extracts), Arg-specific regulation is always
observed.
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Fig. 5.
Effects of Arg concentration on Arg-specific
regulation. A, effects of arg-2 AAP-LUC
fusion in N. crassa translation extracts containing varying
concentrations of Arg assayed by toeprinting. Equal amounts of
synthetic RNA transcripts (120 ng) were translated in reaction mixtures
that contained 10, 150, 500, 2000, or 5000 µM Arg and a
10 µM concentration of each of the other 19 amino acids.
Transcripts were analyzed by toeprinting as described in the
legend to Fig. 2. The transcripts encoded either the wild-type
(wt) or the D12N mutant arg-2 AAP as a uORF in
the 5' leader. The arrow indicates the position of toeprint
products corresponding to ribosomes bound at the AAP initiation codon
(AUGAAP). The arrowhead indicates the
position of toeprint products corresponding to ribosomes stalled at the
codon immediately following the last codon of the AAP in N. crassa translation extracts containing high Arg concentrations.
The bracket indicates the positions of toeprint products
corresponding to ribosomes stalled in the luciferase coding region in
N. crassa translation extracts containing high Arg
concentrations. Dideoxynucleotide sequencing reactions for the
wild-type arg-2 AAP-LUC fusion template are shown on the
left; the nucleotide complementary to the dideoxynucleotide
added to each sequencing reaction is indicated below the
corresponding lane so that the sequence of the template can be directly
deduced; the 5'-to-3' sequence reads from top to bottom. B,
effects of arg-2 AAP-LUC fusion on Arg-specific regulation
in translation extracts derived from S. cerevisiae, N. crassa, and wheat germ assayed by measuring luciferase enzyme
activity. Equal amounts (12 ng) of arg-2 AAP-LUC fusion RNA
were translated in S. cerevisiae, N. crassa, and wheat germ
extracts containing 10, 150, 500, 2000, or 5000 µM Arg
and a 10 µM concentration of each of the other 19 amino
acids. Mean values and standard deviations from measuring the firefly
luciferase enzyme activity in two independent translation reactions are
given. Activities of sea pansy luciferase translated from a second RNA
encoding this enzyme (an RNA lacking AAP regulatory sequences that was
included as an internal control in each reaction mixture) are indicated
below the corresponding firefly luciferase activities.
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The level of charged arginyl-tRNA might be a signal for Arg-specific
regulation mediated by the AAP. Transcriptional attenuation of the
amino acid biosynthetic operons in bacteria is modulated by the level
of charged tRNA (20). The charging status of tRNA controls the
translation of Gcn4p in yeast (19). To determine whether the levels of
aminoacylation of arginyl-tRNAs change when different Arg
concentrations are present in translation extracts, we adapted a method
that has been successful in determining the levels of aminoacylation of
tRNAs in vivo (24). Reaction mixtures were supplemented with
increasing concentrations of Arg and incubated for 10 min. Then total
nucleic acid was obtained under conditions in which tRNA charging is
maintained and the tRNAs separated by polyacrylamide gel
electrophoresis using conditions that resolve charged and uncharged
tRNAs. The charging status of different tRNA species were detected by
Northern blot hybridization using 32P-labeled
oligonucleotide probes complementary to the specific tRNAs of interest.
We first checked the charging status of S. cerevisiae tRNAs
in yeast extracts with a positive control
tRNAiMet probe. Predominantly uncharged
tRNA is observed after alkali treatment of the tRNAs isolated from
extracts (Fig. 6A,
lane 1). tRNA is mostly uncharged in
T0 S. cerevisiae extracts, which have not been
incubated and which have not been supplied with an energy regeneration
system or additional amino acids (Fig. 6A, lane
2). In contrast, in complete translation extracts containing
10, 150, 500, or 2000 µM Arg and 10 µM each
of the other 19 amino acids that have been incubated for 10 min, the
tRNAiMet is predominantly fully charged
(Fig. 6A, lanes 3-6).
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Fig. 6.
Charging status of tRNAs in S. cerevisiae and wheat germ extracts. Charged
(aminoacylated) tRNAs (filled arrowheads) and
uncharged (deacylated) tRNAs (open arrowheads)
were separated on acid-urea-polyacrylamide gels followed by
electrophoretic transfer to Nytran Plus membranes. Specific tRNA
species were detected by Northern blot hybridization (24) using
5'-32P-labeled oligonucleotides complementary to specific
regions of the tRNA species that are indicated at the bottom of each
panel. For each panel, lane 1 contains
alkali-treated tRNAs (deacylated tRNAs) from the extracts indicated;
lane 2 contains tRNAs in extracts at time 0 (T0 extract); lanes 3-6 show tRNAs
in translation reaction mixtures after 10 min of incubation; reaction
mixtures contained 10, 150, 500, and 2000 µM Arg,
respectively, and 10 µM each of the other 19 amino
acids.
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The arginyl-tRNAs detected with probes that should recognize three
different tRNAArg species showed the same pattern of
charging as the tRNAiMet control (Fig.
6, B and C). The arginyl-tRNAs were mainly
uncharged in T0 extracts but maximally charged at even the
lowest concentration of Arg added to extracts (10 µM).
Thus, in S. cerevisiae extracts, the charging status of
arginyl-tRNAs did not appear to change in response to levels of Arg
supplement that result in Arg-specific translational regulation.
Similar studies were attempted using N. crassa extracts.
Both tRNAiMet and
tRNAGAAPhe were maximally charged under
normal translation conditions (data not shown). However, we were unable
to detect N. crassa tRNAArg with S. cerevisiae probes, and lacking N. crassa
tRNAArg sequences to design specific probes, we were unable
to determine the charging status of these tRNAs.
Since AAP-mediated Arg-specific regulation was observed in wheat germ
extracts (Fig. 5B), we analyzed the charging status of
methionyl- and arginyl-tRNAs in these extracts (Fig. 6,
D-F). The results were similar to those obtained with yeast
extracts except that wheat germ tRNAs were already charged in
T0 extracts. The reason for this difference between wheat
germ and yeast extracts was not determined, but it might reflect the
presence of a high level of ATP in T0 wheat germ extracts,
since ATP and an ATP regenerating system are present in T0
wheat germ extracts but not T0 yeast extracts.
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DISCUSSION |
The S. cerevisiae Arg biosynthetic gene CPA1
contains a cis-acting control region functionally analogous
to a bacterial operator in repressing gene expression in response to
Arg (35). Regulation is indicated to act at the level of translation
because mutations causing constitutive expression affect the
translation of a peptide encoded in the 5'-leader of the
CPA1 mRNA (5) that is evolutionarily conserved (Fig.
1A). We examined the role of this AAP in Arg-specific regulation by programming translation extracts from S. cerevisiae, N. crassa, and wheat germ with mRNA
containing the AAP and firefly LUC reporter sequences. Using fungal
extracts, in which the movement of ribosomes could be examined by
primer extension inhibition, the wild-type but not mutant
CPA1 and arg-2 AAPs acted similarly to stall the
movement of ribosomes immediately after AAP translation. Regulation did
not appear to be a response to the level of charged Arg-tRNA. The
observation that tRNAs were maximally charged in extracts provided with
10 µM exogenously supplied amino acids is consistent with
the observation that this amount of supplement is sufficient for near
maximal translational activity and consistent with the observed
Km of 1.5 µM for Arg of the
purified S. cerevisiae arginyl-tRNA synthetase (36).
The absence of an apparent role for the level of charged tRNA in a case
of translational regulation of an amino acid biosynthetic gene is
unprecedented. tRNA charging is important for the transcriptional attenuation of amino acid biosynthetic genes in prokaryotes, in which
lack of specific charged tRNAs causes critical stalls in the
translation of upstream leader peptides (20), and in the translational
regulation of S. cerevisiae GCN4 through the
GCN2-encoded kinase, which is activated by binding to
uncharged tRNA (37). However, these latter control mechanisms that
respond to tRNA charging are designed to respond to amino acid
limitation. CPA1 in fact responds to Arg limitation through
a GCN4-mediated process (38), and N. crassa arg-2
responds to amino acid limitation through a cpc-1 mediated
process (9, 39, 40). cpc-1 is the homolog of GCN4
(41, 42), and the translation of its mRNA is also regulated by
amino acid limitation (7). The available evidence concerning the signal
for the CPC-1-mediated response to amino acid limitation in N. crassa indicates that it is uncharged tRNA (42), as it is for the
Gcn4p-mediated response in yeast. Strikingly, N. crassa
contains a close homolog of GCN2 known as cpc-3,
and cpc-3 mutants have phenotypes similar to gcn2
mutants (43). Thus, it appears that CPA1 and
arg-2 share both a conserved mechanism to respond to amino
acid limitation (through GCN4/GCN2 and
cpc-1/cpc-3, respectively) and a conserved mechanism to
respond to Arg surplus (through translation of the
cis-acting AAP). The response to limitation appears mediated
by the level of tRNA; the response to surplus appears to be mediated
differently. Because fungi store large amounts of Arg in the vacuole
(44) (the concentration of Arg in the vacuole of S. cerevisiae grown in Arg-containing medium is 430 mM
(45) and a high concentration of Arg is also stored in the vacuole of
N. crassa (46), it would seem logical that they possess a
regulatory mechanism to modulate Arg biosynthesis in response to
cytosolic concentrations of Arg far exceeding those necessary for the
charging of tRNA.
The S. cerevisiae and N. crassa AAPs exerted
regulatory effects on translation in plant as well as fungal systems.
These data provide constraints for models of how Arg exerts its
regulatory effect. Presuming that Arg, or a close metabolite, is
directly responsible for regulation, then there are at least three ways that it could function to control the movement of ribosomes. High concentrations of Arg could result in modification of the translational machinery (analogous to uncharged tRNA resulting in eIF2
phosphorylation). This modified machinery would then be sensitive to
stalling by the wild-type AAP. Wheat germ and fungal systems might
share regulatory pathways (or have independently derived regulatory
pathways) that enable AAP-mediated Arg-specific control to be observed
in vitro.
Second, Arg might not cause modification of a translational component,
but instead might interact directly with the translational machinery,
causing the machinery to become sensitive to AAP-mediated stalling. In
addition to the well established interaction of Arg with regulatory
proteins such as the E. coli Arg repressor (e.g. Ref. 47), Arg can also interact with RNA. In the case of the Tetrahymena rRNA self-splicing intron, Arg competes for GTP
binding (48). The human immunodeficiency virus trans-acting responsive element RNA binds an Arg residue of Tat; it also binds the free amino
acid, blocking the interaction of the RNA with Tat (49). RNA aptamers
can also be selected on the basis of their Arg binding (50). There is
already precedent for the direct inhibition of ribosomal peptidyl
transferase activity by Arg (51). However, that inhibitory effect was
elicited by either D-Arg or L-Arg, but
D-Arg does not elicit AAP-mediated control in N. crassa extracts (15) or S. cerevisiae extracts (data
not shown).
Finally, Arg might exert its effect by interacting directly with the
AAP. The AAP-Arg complex would stall the ribosome, and thus the AAP
would function as a cis-acting "argometer" from within the ribosome. This is possibly the simplest model consistent with the
data available thus far, but there is yet no direct evidence supporting
it relative to the other models.
That ribosomes which have translated the AAP are sensitive to stalling
by Arg in extracts is clear. This effect could explain the
translational response to Arg observed in vivo in N. crassa, in which Arg reduces the average number of ribosomes
associated with arg-2 mRNA (7). But, in addition to
reduced translation, the steady-state level of N. crassa
arg-2 mRNA is also reduced by growth in Arg (7-9, 39, 52).
Similarly, Arg affects the level of CPA1 transcript (53).
Could there be a role for the uORF-encoded AAP in regulating the level
of transcript in response to Arg in these systems, perhaps as a
consequence of its function to modulate ribosome stalling? In N. crassa continuously grown in the presence of Arg, a reporter gene
containing the wild-type arg-2 uORF shows a reduction in
both the level of translation and the level of mRNA, as does the
endogenous arg-2 gene. Introduction of the D12N mutation
into the uORF of the reporter gene causes loss of regulation at both
translation and mRNA levels in vivo, while the
endogenous arg-2 gene remains regulated (9). The wild-type
CPA1 mRNA is known to be destabilized by growth in Arg (53). One hypothesis, which remains to be tested, that could link our
observations on stalling in vitro in S. cerevisiae and N. crassa systems with observations
in vivo in these fungi on regulation at the level of
mRNA is that ribosome stalling at the wild-type AAP termination
codon in response to Arg triggers RNA destabilization. Consistent with
this possibility, links between uORF termination codons and RNA
stability are observed in S. cerevisiae (54, 55).
In summary, translation of the evolutionarily conserved AAP in the
presence of high concentrations of Arg causes ribosomes to stall. In
S. cerevisiae, N. crassa, and other fungi, the
AAP is encoded by a uORF in the 5'-leader of the transcript. The data are consistent with a model for regulation in which the AAP-mediated stalling of ribosomes at the uORF termination codon in response to Arg
blocks downstream initiation. Another uORF whose sequence is
evolutionarily conserved, the second uORF of cytomegalovirus gpUL4
(gp48) (56), also causes ribosomes to stall after they have translated
it (57, 58). The existence of other uORFs whose peptide sequences are
known to be important for regulation (2), such as the uORF in
S-adenosylmethionine decarboxylase (59), as well as the
existence of evolutionarily conserved uORFs of unknown regulatory
function such as are present in transcripts specifying mammalian
HER2/neu (10), bcl-2 (11, 12),
CCAAT/enhancer-binding protein (13), and plant bZIP proteins (60),
suggest that other conserved uORF-encoded peptides may prove to have
special roles in regulating translation.
§,
**
TOP
ABSTRACT
INTRODUCTION
