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J. Biol. Chem., Vol. 275, Issue 35, 26710-26719, September 1, 2000
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From the
Received for publication, April 13, 2000, and in revised form, May 17, 2000
Neurospora crassa arg-2 mRNA
contains an evolutionarily conserved upstream open reading frame (uORF)
encoding the Arg attenuator peptide (AAP) that confers negative
translational regulation in response to Arg. We examined the regulatory
role of the AAP and the RNA encoding it using an N. crassa
cell-free translation system. AAPs encoded by uORFs in four fungal
mRNAs each conferred negative regulation in response to Arg by
causing ribosome stalling at the uORF termination codon. Deleting the
AAP non-conserved N terminus did not impair regulation, but deletions
extending into the conserved region eliminated it. Introducing many
silent mutations into a functional AAP coding region did not eliminate
regulation, but a single additional nucleotide change altering the
conserved AAP sequence abolished regulation. Therefore, the conserved
peptide sequence, but not the mRNA sequence, appeared responsible
for regulation. AAP extension at its C terminus resulted in
Arg-mediated ribosomal stalling during translational elongation within
the extended region and during termination. Comparison of Arg-mediated stalling at a rare or common codon revealed more stalling at the rare
codon. These data indicate that the highly evolutionarily conserved
peptide core functions within the ribosome to cause stalling;
translational events at a potential stall site can influence the extent
of stalling there.
The fungi contain two carbamoyl-phosphate synthetases
(CPS)1 (1). CPS-P produces
carbamoyl phosphate for the synthesis of pyrimidines and CPS-A produces
carbamoyl phosphate for the synthesis of Arg. The Neurospora
crassa arg-2 and Saccharomyces cerevisiae CPA1 genes
encoding the CPS-A small subunit are subject to a unique form of
Arg-specific negative translational regulation that requires a
conserved cis-acting peptide coding region present as an
upstream open reading frame (uORF) in the 5'-leader of each transcript (2-6). Translational regulation by eukaryotic uORFs is becoming an
increasingly well documented form of genetic control (7-9), but there
is little understanding of the mechanistic basis for uORF function in
most cases.
The roles of the arg-2 and CPA1 uORFs in
translational control have been investigated using cell-free
translation extracts from N. crassa and S. cerevisiae in which Arg-specific regulation mediated by the AAP is
reconstituted. When either uORF is placed in the 5'-leader of capped
and polyadenylated synthetic RNA transcripts encoding firefly
luciferase (LUC), synthesis of the LUC is reduced when extracts contain
a high concentration of Arg (see Ref. 10, and references therein). The
positions of ribosomes at rate-limiting steps in translation have been
assessed using a primer-extension inhibition (toeprint) assay; Arg
causes ribosomes to stall at the arg-2 and CPA1
uORF termination codons in each extract (10, 11). As a consequence of
stalling, fewer ribosomes initiate translation at the downstream
initiation codon; the stalled ribosomes appear to block ribosomes
engaged in scanning (11, 12). However, although ribosomes normally
stall at these uORF termination codons, when the uORF coding regions
are fused directly to LUC, ribosomes also stall during elongation in
the region immediately downstream of these coding regions (10, 13). The
regulatory effects of Arg appear independent of the extent of
arginyl-tRNA charging because the tRNAs appear maximally charged at
concentrations of Arg substantially below those that exert regulatory
effects (10).
The product of the arg-2 and CPA1 uORFs has been
named the Arg attenuator peptide (AAP) because of its function in
translational regulation (10, 13). Similar peptides are encoded by
uORFs in the transcripts of the corresponding genes of three other
euascomycetes, Magnaporthe grisea, Trichoderma
virens, and Aspergillus nidulans (Fig. 1). Comparisons
of these uORF-encoded peptides indicate that the central amino acid
sequence and the overall length are highly conserved (Fig. 1). The
function of the M. grisea, T. virens, and
A. nidulans uORFs has not been tested. Naturally occurring polymorphisms observed in the second uORF of the cytomegalovirus UL4
transcript, whose peptide sequence is important for it to function to
stall ribosomes, can result in the loss of ability to control
translation (14). Because of this, and because the Schizosaccharomyces pombe carbamoyl-phosphate synthetase
small subunit gene (SPBC56F2.09C) apparently lacks a uORF, it is
important to determine whether these other fungal uORFs function in regulation.
Mutations in the N. crassa and S. cerevisiae AAPs
that alter amino acid residues in the conserved central region
eliminate regulation (2, 5, 6, 10, 11, 15, 16). However, although a
primary role for the AAP coding sequence is indicated, it has not been
established whether the sequence of the RNA also has a regulatory role
outside of its capacity to encode the AAP. Such a regulatory role needs
to be examined because RNA sequences can bind to Arg directly (17-19).
Furthermore, as in the transcriptional attenuation of bacterial amino
acid biosynthetic operons, specific sequences within the leader
peptide's coding region might form structural elements at the level of
nucleic acid critical for regulation (20). Additionally, it is unclear
whether other features of the AAP coding region, such as its length,
irrespective of coding sequence, are important for regulation.
We examined the role of the fungal uORFs that confer Arg-specific
regulation by investigating the importance of the uORF-encoded peptide
sequence and the RNA sequence encoding the peptide to more fully
understand the requirements for these uORFs' regulatory function.
These data indicate that the most highly conserved features of these
uORF-encoded peptides are both necessary and sufficient to confer
Arg-specific regulation and that the nascent peptide moiety itself, and
not structural features of the RNA encoding it, is responsible for regulation.
DNA Templates and RNA Synthesis--
Plasmids were designed to
produce capped and polyadenylated synthetic RNA encoding firefly LUC
with the wild-type or mutant AAP sequences in the RNA 5'-leader region
(Fig. 2). Most plasmids (Table I) contained mutations introduced by PCR
with mutagenic primers (5, 6) or by megaprimer PCR (21). Others were constructed by fragment-insertion or fragment-replacement using synthetic oligonucleotides (Table II). Oligonucleotides used for plasmid constructions are listed in Table III. Additional plasmids used
were described previously (11, 13, 16).
Plasmid DNA templates were purified by equilibrium centrifugation (16)
or by using the Qiagen Plasmid Midi Prep kit; templates were linearized
with EcoRI. Capped, polyadenylated RNA was synthesized with
T7 RNA polymerase from linearized plasmid DNA templates, and the yield
of RNA was quantified as described (16).
Cell-free Translation of RNA and Primer Extension Inhibition
(Toeprint) Assays--
The reaction conditions for in
vitro translation using N. crassa extracts were as
described (13, 16). Cell-free translation extracts were prepared from
N. crassa as described (16). In some experiments, this
preparation procedure was modified by changing the method of grinding
the cells (22); following harvesting of mycelial pads and rinsing with
buffer A, the mycelial pads were frozen in liquid nitrogen, placed in a
pre-chilled (
The primer extension inhibition (toeprint) assays were accomplished as
described using primer ZW4 (11); 8 µl of sample instead of 4 µl was
loaded onto each gel lane. The gels were dried and exposed to screens
of a Molecular Dynamics PhosphorImager for approximately 24 h. All
toeprint data shown were representative of multiple experiments.
The Conserved Fungal AAPs Function in Arg-specific
Regulation--
The functions of the M. grisea, A. nidulans, and rat uORFs in Arg-specific regulation were tested in
parallel with the N. crassa uORF in the N. crassa
cell-free translation system. The uORF in the rat carbamoyl-phosphate
synthetase gene (23) encodes the peptide (MYRL*), which differs from
the fungal uORF-encoded peptides. Equal amounts of each RNA sample were
translated in reaction mixtures containing low (10 µM) or
high Arg (500 µM); LUC activity and the positions of
ribosomes on each RNA were assayed (Fig. 3).
For each of the fungal uORFs, a high concentration of Arg caused
decreased synthesis of LUC and caused ribosomes to stall at the uORF
termination codon (Fig. 3, compare lanes 2 and
1, lanes 5 and 4, and
lanes 8 and 7; relative LUC activities
are indicated at the top of each pair of lanes). Thus, each of the fungal uORFs acted similarly to cause Arg-specific regulation in the
N. crassa cell-free system. In reaction mixtures containing high Arg that were programmed with RNA containing the N. crassa and M. grisea uORFs, which showed stronger
Arg-specific regulation than reaction mixtures programmed with RNA
containing the A. nidulans uORF, additional toeprint signals
were observed 21-30 nt upstream of the toeprint corresponding to the
termination codon. These signals are considered likely to correspond to
ribosomes stalled behind the ribosomes stalled at the termination codon
(11). High Arg also caused a reduction in the signal corresponding to ribosomes at the N. crassa and M. grisea uORF
initiation codons. This is presumably because the toeprint assay
preferentially detects ribosomes that are closest to the primer-binding
site when multiple ribosomes are present on an mRNA (11).
Comparison of toeprint signals from reaction mixtures programmed with
RNA containing the rat uORF and RNA in which the rat uORF initiation
codon was eliminated revealed ribosomes at the rat uORF's initiation
codon and termination codon (Fig. 3, compare lanes
10 and 11 with lanes 13 and
14). Thus, the rat uORF appeared to be translated. However,
in contrast to the Arg-specific regulation of LUC synthesis and of
ribosome stalling conferred by each of the fungal uORFs, the rat uORF
did not confer Arg-specific regulation (Fig. 3, compare
lanes 11 and 10).
Effects of Deleting the N Terminus of the AAP--
Although the
overall length of the AAP and its central region are evolutionarily
conserved, the sequence of the AAP's N terminus is not conserved (Fig.
1). Possibly, the length of the N
terminus is important, even if the sequence is not, since shortening
the C terminus eliminates function (Ref. 11 and see below). To test this, a series of deletions were constructed to shorten the AAP at its
N terminus. First, a unique NdeI restriction site was
introduced into the N. crassa arg-2 sequence in the region
that included the AAP initiation codon. Introduction of this site
improved the initiation context of the uORF but did not change its
predicted coding sequence (Fig.
2A). Next, a series of
deletions were constructed using PCR and mutagenic primers (Table
I). Regulation exerted by the uORF
containing the NdeI site, and uORFs containing deletions of
amino acid residues 2, 2-3, 2-4, 2-5, 2-6, or 2-7 were compared with regulation mediated by the wild-type uORF and by a uORF in which
Asp-12 was mutated to Asn (D12N). The latter mutation abolishes regulation in vivo (6) and in vitro (10, 11, 13,
16).
Equal amounts of capped and polyadenylated synthetic RNAs representing
each construct were translated in N. crassa extracts (Fig.
4). Relative to a construct
containing the uORF in the wild-type initiation context, regulation by
Arg was increased by introduction of the NdeI site, which
improves the initiation context of the uORF (Fig.
4). Increased initiation at the uORF
start codon was evident from the increased toeprint signal that
corresponded to ribosomes at the uORF initiation codon in the
NdeI construct compared with the wild-type construct in
extracts containing low Arg (Fig. 4, compare lane
5 with lane 1). This is consistent
with the previously observed increases in regulation occurring when the
uORF initiation context is improved by other mutations (16).
Deletion of codon 2 (
The N-terminal region of the AAP could also be extended (Fig.
2C) without abolishing its regulatory function (data not
shown). All of the extended AAPs tested retained regulation based on
LUC and toeprint assays; in contrast, similarly extended AAPs that also
contained the D12N mutation showed no regulatory capacity.
The RNA Sequence Is Not Important for Regulation beyond Its
Capacity to Encode a Functional AAP--
Is the AAP alone required for
regulation, or does the RNA that encodes it also play an additional
regulatory role? To test this possibility, we examined the role of the
AAP-encoding RNA sequence in its entirety by effecting a radical change
in the RNA sequence specifying the functional 20-residue AAP in which residues 2-4 were deleted (
Analyses of reporter gene activity by LUC assay and toeprint assays
(Fig. 5) indicated that the RNA sequence
was not important for regulation beyond its capacity to encode the AAP.
Regulation was apparent for RNA containing only silent mutations in the
AAP coding sequence (Fig. 5, compare lanes 1 and
2 with lanes 6 and 7). A
single additional nucleotide substitution that changed Asp to Asn
eliminated regulation (Fig. 5, lanes 4 and
5). Therefore, the RNA sequence does not appear to have a
regulatory role other than to encode a functional AAP.
Introduction of the silent mutations into the RNA sequence slightly
reduced regulation and the strength of the toeprint signal corresponding to ribosomes at the uORF termination codon (Fig. 5). One
possible reason for this consistent with the LUC activity and primer
extension data is that these substitutions caused changes in the
structure of the mRNA that reduced initiation at the AAP start codon.
Lengthening but Not Shortening the uORF-encoded AAP's C Terminus
Permits Regulation--
The synthesis of a polypeptide containing the
wild-type arg-2 AAP coding sequence fused directly to LUC is
subject to Arg-specific regulation; ribosomes stall at sites
immediately distal to the 24-codon AAP located within the LUC coding
region (experiments described below; Refs. 10 and 13). This suggested
the possibility that the C terminus of the uORF could be extended
without causing loss of regulatory function. To test this possibility,
a series of constructs were made to extend the uORF-encoded N. crassa AAP at its C terminus; the stop codon (normally codon 25)
was moved to positions 26, 27, 28, 29, or 30. As the basis for these
constructs, a plasmid was used in which several unique restriction
sites (AgeI, SpeI, MluI) were
introduced into the AAP coding region to facilitate construction; the
introduction of the AgeI site changed codon 2 from Asn to
Thr and the other mutations were silent mutations (Fig. 2, A
and C). Toeprint signals corresponding to ribosomes stalled
at the uORF termination codon and to additional ribosomes stalled
21-30 nt upstream of ribosomes at the termination codon were similar
in RNAs specifying the wild-type uORF or the mutant uORF containing
AgeI, SpeI, and MluI sites (Fig.
6, compare lanes 13 and 14 with lanes 7 and 8).
A slight increase in regulation was observed with the mutant uORF
relative to the wild-type uORF; increased regulation was associated
with an increased toeprint signal corresponding to ribosomes at the
uORF initiation codon (Fig. 6, compare lanes 13 and 14 with lanes 7 and 8),
consistent with observations on the effect of introducing the
NdeI site into the uORF coding region (Fig. 4) described
above.
Additional controls were analyzed in parallel with the extended uORF
constructs (Fig. 6). Elimination of the uORF initiation codon (
LUC activity and toeprint analyses of the positions of ribosomes on the
extended uORFs revealed a number of striking features. Moving the
termination codon from codon 25 to codon 26, 27, 28, 29, or 30 either
did not affect or slightly increased regulation. The positions of
primer extension products corresponding to the uORF termination codon
(asterisks) and to the initiation codon (arrowheads) appeared in positions exactly as predicted from
the sizes of the extended uORFs. Arg increased the signals
corresponding to ribosomes at uORF termination codons and decreased the
signal corresponding to ribosomes at initiation codons. Arg also
increased the stalling of ribosomes at codon 25 and subsequent sense
codons in the C-terminally extended uORFs independent of its effects at
the uORFs' termination codons, indicating that stalling during elongation occurred as well as stalling during termination.
It was of interest to determine whether the coding region could be
extended to the point at which increased stalling at the termination
codon in response to Arg was lost. In an AAP-LUC fusion construct,
elongating ribosomes stall in a window corresponding to approximately
six codons in the presence of high Arg (Fig. 7, compare lanes 2 and 1). A single nucleotide deletion at codon 25 causes a
frameshift so that the AAP reading frame has a termination codon at
codon 32 (Fig. 2B). Primer-extension analyses of this frameshift construct indicated that, although Arg-regulated stalling of
ribosomes occurred between codons 25 and 30, Arg did not affect stalling of ribosomes at codon 32. An indication of the high precision of the toeprint assay for mapping ribosomes is also evident from this
experiment. As a consequence of the frameshift, the AAP initiation codon is 1 nt closer to the primer used for toeprinting, and this is
observed in the positions of the toeprint signals corresponding to
ribosomes initiating at the start of the AAP in wild-type and frameshifted constructs (Fig. 7, compare arrowheads for
lanes 4 and 2).
An unexpected result was observed in the experiments shown in Fig. 6.
Although toeprint signals are observed in the region 21-30 nt upstream
of the termination codon of functional AAPs when the termination codon
is at its normal position (e.g. Figs. 3-6; see also
additional experiments in Refs. 10, 11, and 13), when the uORF was
extended at its C terminus, these strong upstream signals disappeared.
The explanation for this is unclear. Were these signals to correspond
to ribosomes stalled behind ribosomes stalled at termination codons,
then it might have been expected that they remain in the same position
relative to the termination codon in each of the C-terminally extended constructs.
Arg-specific Stalling during Elongation Is Better Facilitated by a
Rare Codon than a Common Codon--
Does the rate of translation
affect the extent of ribosome stalling at a given site? Rare codons are
considered to cause slowing of translation (see Ref. 24, and references
therein). Therefore, we tested the effect on stalling of placing a rare
codon versus a common codon at the same position in the
mRNA (Fig. 2B) and examined the extent of ribosome
stalling at that codon (Fig. 8). In high
Arg, a construct containing the rarest Leu codon (UUA; 1.7% usage
(Ref. 25)) at codon 25 of the AAP-LUC fusion gene showed greater
stalling at that position than a construct containing the most common
Leu codon at that position (CUC; 42% usage (Ref. 25)) at that position
(Fig. 8, compare lanes 2 and 6). When less stalling was observed at this position, relatively more stalling was observed downstream, and the extent of regulation was similar in
both constructs as determined by LUC assay (Fig. 8). When constructs contained the D12N mutation, regulation by Arg was eliminated (Fig.
8).
The results indicate that the synthesis of a core peptide of
evolutionarily conserved sequence can regulate ribosome stalling on RNA
in response to the concentration of Arg. The sequence of the template
RNA encoding this peptide does not have a discernible active role in
this process although it can affect the extent of stalling and the
positions of stalled ribosomes. The ribosomes that have synthesized the
nascent peptide moiety appear to become transiently sensitive to
stalling by Arg (or a close metabolite) as they translate nearby
downstream RNA sequence. The data suggest this control mechanism is
also used by the fungi M. grisea, T. virens, and
A. nidulans, since the peptides encoded by uORFs in the
corresponding genes of each of these fungi also function to stall
ribosomes in response to Arg in the N. crassa cell-free system.
The rat CPS transcript contains a uORF that is different from the
fungal CPS-A transcript uORFs. Although the rat uORF is recognized by
fungal ribosomes in vitro (Fig. 3), it does not appear to
function to modulate translation in response to Arg. This is consistent
with its lack of sequence similarity, since even minor changes
affecting conserved AAP residues eliminate its regulatory function.
Although the carbamoyl phosphate produced by mammalian CPS I can
be used for Arg biosynthesis (26), and there are indications for
post-transcriptional control mediated by the 5'-leader of the rat CPS
mRNA (27), the regulatory role of this uORF, if any, remains unknown.
Currently, the most thoroughly studied of the uORFs whose encoded
peptide sequences are important for translational control are the
fungal uORFs encoding the AAP, uORF2 of cytomegalovirus UL4, and the
uORF of mammalian S-adenosyl decarboxylase (see Ref. 7, and
references therein). The limited introduction of silent mutations into
the coding regions of the S. cerevisiae CPA1 (3) and
cytomegalovirus UL4 (28) uORFs, as well as the introduction of silent
mutations at all five mutable codons in the mammalian S-adenosyl decarboxylase uORF (29), were consistent with the lack of importance of those RNA sequences for regulation other than
through their capacity to encode a specific peptide sequence. We have
analyzed the regulated stalling of ribosomes mediated by AAPs encoded
by natural S. cerevisiae, M. grisea, A. nidulans, and N. crassa sequences, as well as by a
synthetic AAP coding region in which silent mutations alter over 40%
of its nucleotide sequence (Ref. 10 and this work; see also Refs. 11
and 13). The data indicate that it is highly unlikely that there is a
role for the RNA in mediating Arg-regulated ribosome stalling outside of its capacity to encode the AAP. Alignment of the wild-type fungal
AAP coding sequences and the shortened, multiply substituted functional
N. crassa AAP coding sequence shows no more than two consecutive nucleotides are conserved among all of the sequences, except for the Trp codon, TGG. Furthermore, we have found that this
codon can be changed to a Tyr codon (TAC) and regulation maintained.2 Thus, if the RNA
sequences encoding AAPs were important outside of their coding roles,
then only a very limited primary conserved sequence must be important.
Such a limited sequence might include an Arg codon because analyses of
RNA aptamers that bind Arg indicate that an Arg codon is often part of
the binding site (17). However, for the fungal AAPs, no Arg codons,
either in the AAP reading frame or in alternative reading frames, are
conserved among all of the sequences (data not shown).
In addition to providing insight into the requirements for the role of
a nascent peptide in translational regulation, these results indicate
that rare codons can cause conditional effects in translation that
depend on other contributing factors. Although very little stalling of
ribosomes occurred when either a rare Leu codon or a common Leu codon
were present at codon 25 in an AAP-LUC construct at a low concentration
of Arg, the rare codon enabled substantially more stalling than a
common codon at a high concentration of Arg (Fig. 8). These results
indicate that, although a termination codon is not required for
Arg-specific ribosome stalling after synthesis of the AAP, a slow step
in translation such as an encounter with a stop codon or a rare codon
facilitates stalling. Such a step may provide more time or a more
favorable environment for the nascent AAP to exert its regulatory function.
The Arg-specific control of ribosome movement mediated by fungal AAPs
so far represents a unique form of regulatory control in response to an
amino acid. Related small molecules (polyamines) cause the
translational regulation of mammalian S-adenosylmethionine decarboxylase via the action of a uORF whose sequence is critical for
control (30, 31). It will be interesting to determine whether the
control mechanisms are similar. The amino acid methionine is involved
in the post-transcriptional regulation of the first enzyme committed to
Arabidopsis methionine biosynthesis, cystathionine The stalling of ribosomes during translation is commonly thought to be
mediated by secondary structures in the mRNA or by encounters with
rare codons; nascent peptides have also been implicated in ribosome
stalling in some instances (see Refs. 34 and 35, and references
therein). Nascent peptides encoded by uORFs in eukaryotic and
prokaryotic transcripts also are implicated in ribosome stalling (7,
36). The data presented here provide the strongest evidence to date
that a eukaryotic peptide can cause the regulated stalling of ribosomes
involved in both termination and elongation, and that, although
mRNA structure or the presence of rare codons may influence the
extent of stalling, it is the nascent peptide itself that is primarily
responsible for controlling the subsequent movement of the ribosome.
We thank Anthony Gaba and Alan Sachs for
critical reading of the manuscript.
*
This work was supported by National Institutes of Health
Grant GM47498.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Current address: Howard Hughes Medical Inst., University of
California, Berkeley, CA 94720-3202.
Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.M003175200
2
C. Spevak, P. Fang, and M. S. Sachs,
unpublished data.
The abbreviations used are:
CPS, carbamoyl-phosphate synthetase;
uORF, upstream open reading frame;
AAP, Arg attenuator peptide;
LUC, luciferase;
nt, nucleotide(s);
wt, wild
type;
PCR, polymerase chain reaction.
Evolutionarily Conserved Features of the Arginine Attenuator
Peptide Provide the Necessary Requirements for Its Function in
Translational Regulation*
,§, and¶
Department of Biochemistry and Molecular
Biology, Oregon Graduate Institute of Science & Technology, Beaverton,
Oregon 97006-8921 and the ¶ Department of Molecular Microbiology
and Immunology, Oregon Health Sciences University,
Portland, Oregon 97201-3098
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Firefly LUC constructs made by PCR or megaprimer PCR (MP)a
Firefly LUC constructs made by oligonucleotide insertion
70 °C) mortar, and then powdered with a pestle in the
presence of liquid nitrogen until a fine paste was obtained. The paste
was then transferred into a 50-ml polysulfone Oak Ridge centrifuge tube
(Nalgene) and thawed on ice before centrifugation. This modification
improved the activity of extracts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 1.
Comparisons of the AAPs from N. crassa (N.c., Ref. 37), M. grisea
(M.g., Ref. 38), T. virens
(T.v., Ref. 39), A. nidulans
(A.n., GenBankTM
AJ224085), and S. cerevisiae (S.c.,
Ref. 2). Residues conserved in at least three AAPs are
boxed.
View larger version (34K):
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Fig. 2.
The 5' leader regions and AAP sequences of
arg-2-LUC genes used in this study. A,
sequences of wild-type and mutant templates in which the AAP is encoded
as a uORF. The sequence shown begins with the T7 RNA polymerase-binding
site and ends within the LUC coding region. The amino acid sequences of
the arg-2 AAP and the N terminus of LUC are indicated.
Mutations that result in new restriction enzyme sites are shown below
the wild-type sequence, as is the D12N mutation, which eliminates
regulation. The multiple silent mutations that change the RNA sequence
but not the peptide coding sequence in a shortened AAP are indicated in
lowercase below the wild-type sequence; short
hyphens indicate deleted nucleotide sequence in this
construct. 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.
B, sequences of templates containing wild-type and mutant
AAP-LUC fusion genes. The sequence shown begins with the T7 RNA
polymerase-binding site and ends within the LUC coding region; the
amino acid sequence of the N terminus of the AAP-LUC fusion polypeptide
is indicated. Point mutations are shown below the wild-type sequence.
The ( 
) mutation improves the initiation context for translation and
the D12N mutation eliminates Arg-specific regulation. Common (CTC) or
rare (TTA) Leu codons were inserted at codon 25 of the polypeptide as
indicated (the wild-type peptide lacks a Leu codon at this position, as
indicated by the dashes). The 1-nt deletion within codon 25 that causes a frameshift (indicated fs) results in a
predicted reading frame for a 31-residue peptide; the sequence of the
frameshifted region of this polypeptide is indicated in
lowercase letters above the sequence of the
wild-type peptide. C, peptide sequences of additional mutant
AAPs used in this study. Sequences between ellipses match the wild-type
sequence. Plasmids and oligonucleotides are described in Tables
I-III.
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Fig. 3.
In vitro translation in N. crassa extracts of mRNAs containing uORFs. The
transcripts examined are indicated at the top; the ratio of
LUC activity produced after 30 min in reaction mixtures containing 10 µM Arg versus 500 µM Arg are
also indicated at the top. Equal amounts of synthetic RNA
transcripts (120 ng) were translated in 20-µl reaction mixtures at
25 °C. Reaction mixtures contained 10 µM ( ) or 500 µM (+) Arg and 10 µM each of the other 19 amino acids. After 20 min of translation, 3 µl of the translation
mixtures were toeprinted with primer ZW4 and analyzed next to
dideoxynucleotide sequencing of the wt-
47 construct (pR1031, Table
I; Ref. 11). The nucleotide complementary to the dideoxynucleotide
added to each sequencing reaction is indicated above the corresponding
lane so that the sequence of the template can be directly deduced; the
5'-to-3' sequence reads from top to bottom. The products obtained from
primer extension of each RNA (120 ng) in the absence of translation
reaction mixture (EXT; lanes 3,
6, 9, 12, and 15) and from
translation reaction mixture not programmed with RNA (RNA,
lane 16) are shown for comparison. The
arrowheads indicate the position of the premature
transcription termination products corresponding to ribosomes bound at
the uORF initiation codon. The asterisks indicate the
positions of premature transcription termination products corresponding
to ribosomes at the uORF termination codon.
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Fig. 4.
Effects of deletion of the amino acid
residues at the uORF N terminus on Arg-specific regulation. The
constructs examined (Fig. 2C) are indicated at the
top; LUC activity measurements and reaction conditions were
as described in Fig. 3. Dideoxynucleotide sequencing reactions for the
template encoding the wild-type AAP with the NdeI site
(pR401, Table I) are on the left. The products of a reaction
obtained from primer extension of pure NdeI vector RNA (120 ng) in the absence of translation reaction mixture ( EXT,
lane 19) are shown for comparison. Arrowheads and
asterisks are as for Fig. 3; open
arrowheads indicate ribosomes at the LUC initiation
codon.
N), codons 2 and 3 (NG), or codons 2-4
(NGR) did not substantially affect regulation based on LUC synthesis
or ribosome stalling at the uORF termination codon (Fig. 4). In
contrast, deletion of codons 2-5 (NGRP) reduced regulation based on
these criteria, and deletion of codons 2-6 (NGRPS) or codons 2-7
(NGRPSV) eliminated regulation, as did the D12N mutation (Fig. 4).
Since codon 5 of the N. crassa AAP defines the beginning of
the region in which all of the AAP sequences obtained to date are
conserved (Fig. 1), it appears that the regulatory function of the AAP
is maintained with deletion of the N terminus up to but not including
the conserved region. Therefore, the results indicate that the
nonconserved N-terminal segment of the AAP is dispensable for regulation.
NGR). The coding region for this
shortened AAP was chemically synthesized with 26 of 63 possible base
substitutions incorporated, all of them silent (Fig. 2A). At
least one nucleotide was changed in every codon, except for the
Trp-codon, which cannot tolerate change, and the Asn codon near the C
terminus, which formed part of the MluI site used for
cloning. A 27th mutation was introduced in a second construct, in
addition to the 26 silent mutations, which changed the conserved Asp to
Asn; this latter mutation is predicted to eliminate regulation. We
reasoned that, if the RNA sequence were not important beyond its
capacity to encode the AAP, then the coding region in which over 40%
of the nucleotides have been silently substituted should confer Arg
regulation, although the construct containing these silent mutations
and one additional nucleotide substitution to change the important Asp residue to Asn should not.
View larger version (101K):
[in a new window]
Fig. 5.
Effects of silent mutations in the RNA
sequence encoding a shortened, functional AAP
( NGR) on Arg-specific regulation. The
constructs examined (wt silent, pRER101; D12N silent, pRER102; wt,
pR404) are indicated at the top; LUC activity measurements
and reaction conditions were as described in Fig. 3. Dideoxynucleotide
sequencing reactions for the template containing the wild-type AAP with
silent mutations (pRER101) are on the left. The products
obtained from primer extension of wild-type RNA containing silent
mutations (120 ng) and wild-type RNA (120 ng) in the absence of
translation reaction mixture (EXT; lanes
3 and 8, respectively), and from translation
reaction mixture not programmed with RNA (RNA;
lane 9) are shown for comparison. The
arrowheads indicate the position of the premature
transcription termination products corresponding to ribosomes bound at
the AAP initiation codon. Arrowheads, asterisks,
and open arrowheads are as for Fig. 4.
View larger version (98K):
[in a new window]
Fig. 6.
Effects of addition of the amino acid
residues at the AAP C terminus on Arg-specific regulation. The
transcripts examined (Fig. 2C) are indicated at the
top; LUC activity measurements and reaction conditions were
as described in Fig. 3. Dideoxynucleotide sequencing reactions for the
template encoding an AAP with the termination codon at codon 30 are on
the right. The products obtained from primer extension of
each pure RNA (120 ng) in the absence of translation reaction mixture
( EXT; lanes 3, 6,
9, 12, 15, 18,
21, 24, 27, and 30) and
from translation reaction mixture not programmed with RNA (RNA;
lane 31) are shown for comparison.
Arrowheads and asterisks are as for Fig. 3.
Oligonucleotides used
AUG)
eliminated regulation and all translation-specific toeprint signals in
the uORF coding region (Fig. 6, lanes 1 and 2). The D12N mutation eliminated regulation and all
Arg-specific translational effects on toeprint signals (Fig. 6,
lanes 4 and 5). Shortening the AAP
coding region by changing Ala-24 to a stop codon (A24*) eliminated
regulation and shifted the toeprint corresponding to the termination
codon by one codon, as expected (Fig. 6, lanes 10 and 11). The results of these control experiments were all consistent with previous observations (11).
View larger version (83K):
[in a new window]
Fig. 7.
Ribosome stalling in an AAP-LUC fusion
construct and a frameshifted AAP-LUC fusion construct. The
transcripts examined (Fig. 2B) are indicated at the
top; transcripts were translated in reaction mixtures as
described in Fig. 3. Dideoxynucleotide sequencing reactions for the
template encoding the frameshifted AAP-LUC fusion peptide (Fig.
2B) are on the right. The products obtained from
primer extension of RNA encoding the frameshifted AAP-LUC fusion
peptide (120 ng) in the absence of translation reaction mixture
( EXT; lane 5) are shown for
comparison. The arrowheads indicate the position of the
premature transcription termination products corresponding to ribosomes
bound at the AAP initiation codon. The closed
circles indicate the position of premature termination
products corresponding to ribosomes stalled in the presence of high Arg
at the 25th codon immediately following the 24 codons of the AAP. The
brackets indicate the position of premature termination
products corresponding to ribosomes stalled in the presence of high Arg
at the codons after the 25th codon. The asterisks indicate
the position of ribosomes at the termination codon (codon 32).
View larger version (118K):
[in a new window]
Fig. 8.
Effects of 25th rare versus
common Leu codon on Arg-specific regulation of AAP-LUC fusion
constructs. The transcripts examined (Fig. 2B) are
indicated at the top. LUC activity measurements and reaction
conditions were as described in Fig. 3. The transcripts encoded the wt
AAP-LUC fusion or the D12N AAP-LUC in improved initiation contexts.
Dideoxynucleotide sequencing reactions for the template containing the
wild-type AAP-LUC fusion with rare Leu codon at its 25th position are
on the left. The products obtained from primer extension of
pure AAP-LUC RNA (120 ng) in the absence of translation reaction
mixture ( EXT, lane 13) and from
translation reaction mixture not programmed with RNA (RNA,
lane 14) are shown for comparison. The
arrowheads indicate the position of the premature
transcription termination products corresponding to ribosomes bound at
the AAP initiation codon. The closed circles
indicate the position of premature termination products corresponding
to ribosomes stalled in the presence of high Arg at the 25th codon
immediately following the 24 codons of the AAP. The brackets
indicate the position of premature termination products corresponding
to ribosomes stalled in the presence of high Arg at the codons after
the 25th codon of the peptide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-synthase. A high level of methionine reduces the level of this mRNA; an evolutionarily conserved sequence within the cystathionine -synthase coding region is required for regulation (32). The mechanism remains to be determined. In mammalian systems, Arg is
observed to affect the post-transcriptional control of the level of an
mRNA specifying a cationic amino acid transporter; this mechanism
appears to involve sequences in the 3'-untranslated region of the
mRNA (33).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Oregon Graduate Inst. of Science & Technology, 20000 N.W. Walker Rd., Beaverton, OR 97006-8921. Tel.:
503-748-1487; Fax: 503-748-1464; E-mail:
msachs@bmb.ogi.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Davis, R. H.
(1986)
Microbiol. Rev.
50,
280-313
2.
Werner, M.,
Feller, A.,
Messenguy, F.,
and Piérard, A.
(1987)
Cell
49,
805-813
3.
Delbecq, P.,
Werner, M.,
Feller, A.,
Filipkowski, R. K.,
Messenguy, F.,
and Piérard, A.
(1994)
Mol. Cell. Biol.
14,
2378-2390
4.
Luo, Z.,
Freitag, M.,
and Sachs, M. S.
(1995)
Mol. Cell. Biol.
15,
5235-5245
5.
Luo, Z.,
and Sachs, M. S.
(1996)
J. Bacteriol.
178,
2172-2177
6.
Freitag, M.,
Dighde, N.,
and Sachs, M. S.
(1996)
Genetics
142,
117-127
7.
Geballe, A. P.,
and Sachs, M. S.
(2000)
in
Translational control 2
(Hershey, J. W. B.
, Mathews, M. B.
, and Sonenberg, N., eds)
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, still in press
8.
Schluter, G.,
Boinska, D.,
and Nieman-Seyde, S. C.
(2000)
Biochem. Biophys. Res. Commun.
268,
255-261
9.
Sarrazin, S.,
Starck, J.,
Gonnet, C.,
Doubeikovski, A.,
Melet, F.,
and Morle, F.
(2000)
Mol. Cell. Biol.
20,
2959-2969
10.
Wang, Z.,
Gaba, A.,
and Sachs, M. S.
(1999)
J. Biol. Chem.
274,
37565-37574
11.
Wang, Z.,
and Sachs, M. S.
(1997)
Mol. Cell. Biol.
17,
4904-4913
12.
Kozak, M.
(1999)
Gene (Amst.)
234,
187-208
13.
Wang, Z.,
Fang, P.,
and Sachs, M. S.
(1998)
Mol. Cell. Biol.
18,
7528-7536
14.
Alderete, J. P.,
Jarrahian, S.,
and Geballe, A. P.
(1999)
J. Virol.
73,
8330-8337
15.
Werner, M.,
Feller, A.,
Delbecq, P.,
and Pierard, A.
(1990)
in
Post-transcriptional Control of Gene Expression
(McCarthy, J. E. G.
, and Tuite, M. F., eds), Vol. H49
, pp. 337-346, Springer-Verlag, Berlin
16.
Wang, Z.,
and Sachs, M. S.
(1997)
J. Biol. Chem.
272,
255-261
17.
Yarus, M.
(1998)
J. Mol. Evol.
47,
109-117
18.
Geiger, A.,
Burgstaller, P.,
von der Eltz, H.,
Roeder, A.,
and Famulok, M.
(1996)
Nucleic Acids Res.
24,
1029-1036
19.
Puglisi, J. D.,
Tan, R.,
Calnan, B. J.,
Frankel, A. D.,
and Williamson, J. R.
(1992)
Science
257,
76-80
20.
Yanofsky, C.
(2000)
J. Bacteriol.
182,
1-8
21.
Sarkar, G.,
and Sommer, S. S.
(1990)
BioTechniques
8,
404-407
22.
Otero, L. J.,
Ashe, M. P.,
and Sachs, A. B.
(1999)
EMBO J.
18,
3153-3163
23.
Nyunoya, H.,
Broglie, K. E.,
Widgren, E. E.,
and Lusty, C. J.
(1985)
J. Biol. Chem.
260,
9346-9356
24.
Komar, A. A.,
Lesnik, T.,
and Reiss, C.
(1999)
FEBS Lett.
462,
387-391
25.
Edelmann, S. E.,
and Staben, C.
(1994)
Exp. Mycol.
18,
70-81
26.
Wu, G.,
and Morris, S. M., Jr.
(1998)
Biochem. J.
336,
1-17
27.
Christoffels, V. M.,
van den Hoff, M. J.,
Moorman, A. F.,
and Lamers, W. H.
(1995)
J. Biol. Chem.
270,
24932-24940
28.
Degnin, C. R.,
Schleiss, M. R.,
Cao, J.,
and Geballe, A. P.
(1993)
J. Virol.
67,
5514-5521
29.
Hill, J. R.,
and Morris, D. R.
(1993)
J. Biol. Chem.
269,
726-731
30.
Ruan, H.,
Shantz, L. M.,
Pegg, A. E.,
and Morris, D. R.
(1996)
J. Biol. Chem.
271,
29576-29582
31.
Mize, G. J.,
Ruan, H.,
Low, J. J.,
and Morris, D. R.
(1998)
J. Biol. Chem.
273,
32500-32505
32.
Chiba, Y.,
Ishikawa, M.,
Kijima, F.,
Tyson, R. H.,
Kim, J.,
Yamamoto, A.,
Nambara, E.,
Leustek, T.,
Wallsgrove, R. M.,
and Naito, S.
(1999)
Science
286,
1371-1374
33.
Aulak, K. S.,
Mishra, R.,
Zhou, L.,
Hyatt, S. L.,
de Jonge, W.,
Lamers, W.,
Snider, M.,
and Hatzoglou, M.
(1999)
J. Biol. Chem.
274,
30424-30432
34.
Crombie, T.,
Swaffield, J. C.,
and Brown, A. J.
(1992)
J. Mol. Biol.
228,
7-12
35.
Tsalkova, T.,
Kramer, G.,
and Hardesty, B.
(1999)
J. Mol. Biol.
286,
71-81
36.
Lovett, P. S.,
and Rogers, E. J.
(1996)
Microbiol. Rev.
60,
366-385
37.
Orbach, M. J.,
Sachs, M. S.,
and Yanofsky, C.
(1990)
J. Biol. Chem.
265,
10981-10987
38.
Shen, W.-C.,
and Ebbole, D. J.
(1997)
Fungal Genet. Biol.
21,
40-49
39.
Baek, J.-M.,
and Kenerley, C. M.
(1998)
Fungal Genet. Biol.
23,
34-44
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