From the
Two lines of evidence indicate the importance of the N-terminal
portion of rhodanese for correct folding of the nascent ribosome-bound
polypeptide. A mutant gene lacking the codons for amino acids
1-23 of the wild-type protein is expressed very efficiently by
coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the
ribosomes is enzymatically inactive. The mutant protein does not
undergo the reaction that is promoted by the bacterial chaperone, DnaJ,
which appears to be essential for folding of ribosome-bound rhodanese
into the native conformation. The effect of DnaJ is monitored by
fluorescence from coumarin cotranslationally incorporated at the N
terminus of nascent rhodanese. Secondly, a synthetic peptide
corresponding to the N-terminal 17 amino acids of the wild-type protein
interferes with the synthesis of wild-type rhodanese but has much less
effect on the synthesis of the N-terminal deletion mutant. The
N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type
rhodanese and blocks the chaperone-mediated release and activation of
ribosome-bound full-length rhodanese polypeptides that accumulate
during in vitro synthesis. The results lead to the hypothesis
that the N-terminal segment of rhodanese is required for its
chaperone-dependent folding on the ribosome.
Involvement of chaperones in the steps by which proteins are
synthesized on ribosomes has been inferred from a number of studies.
Beckmann et al. (1990) showed that hsp 70 was bound to HeLa
cell polysomes in what appeared to be a cotranslational association
with the nascent proteins. Nelson et al. (1992) and more
recently Craig et al. (1994) presented genetic evidence that
indicated a requirement for one of the cytosolic forms of hsp 70 for
protein synthesis in yeast. DnaJ was cross-linked to nascent luciferase
when it had been synthesized to a length of 55 amino acids on wheat
germ ribosomes (Hendrick et al., 1993). Recently, Frydman
et al. (1994) released nascent luciferase from reticulocyte
ribosomes by puromycin and demonstrated GroEL homologs and DnaK
homologs to be present as a complex with the nascent protein. These
studies indicate that at least some of the chaperone-mediated reactions
by which nascent proteins are folded into their native conformation
occur on ribosomes. Hardesty et al. (1993) have reviewed
results indicating that nascent peptides are extended from the peptidyl
transferase center into a sheltered domain or cavity of the large
ribosomal subunit. Eisenstein et al. (1994) have considered
evidence indicating that one or more tunnels occur within the large
ribosomal subunit into which the nascent peptide is extended and in
which folding reactions might take place. However, virtually nothing is
known of how and where within its structure a ribosome might
accommodate chaperones, of the molecular details of how individual
chaperones facilitate protein folding within a ribosome, or of the
apparent interplay between the recognized ribosomal reactions of
protein synthesis and the chaperone-mediated folding reactions.
We
have used bovine rhodanese as a model enzyme to study the synthesis and
chaperone-dependent folding of proteins on ribosomes. Rhodanese is a
33-kDa thiosulfate sulfur transferase (EC 2.8.1.1) that is found in the
matrix of all mitochondria (Westley, 1973). Its enzymatic activity is
easily determined by a sensitive assay system, and the protein itself
is well suited to study the processes by which proteins are folded into
their native conformations. Refolding of rhodanese from the denatured
state has been studied extensively (Horowitz, 1993). In eukaryotic
cells the protein is synthesized on cytoplasmic ribosomes and then
transported into the mitochondrial matrix. With the exception of the
N-terminal methionine, the mature mitochondrial enzyme retains the
entire encoded N-terminal amino acid sequence that is required for
targeting, transport, folding, and activation. The consensus features
for targeting to the mitochondria are apparent in the N-terminal 31
amino acids of the native enzyme. This peptide has seven positive
charges that are embedded in what otherwise is a relatively hydrophobic
sequence. The crystal structure of the protein shows that residues
11-22 form an
Recently, Mendoza and
Horowitz (1994) reported that refolding of denatured rhodanese in the
absence or presence of cpn60 and cpn10 (GroEL and GroES) is modulated
by the 23-amino acid sequence at its N terminus. The renaturation
process was substantially inhibited by a peptide consisting of this
sequence. The N-terminal peptide reduced the extent but not the rate of
the reactivation process but did not prevent the interaction of the
protein with cpn60. This finding and the fact that the peptide inhibits
unassisted refolding suggest that the peptide interacts directly with
the partially refolded protein to inhibit its conversion to the native
conformation.
We have shown that enzymatically active rhodanese can
be formed during synthesis of the protein in a coupled
transcription/translation system that is derived from Escherichia
coli (Tsalkova et al., 1993). However, full-length
rhodanese chains accumulate as peptidyl-tRNA on ribosomes during the
course of synthesis in the cell-free system (Kudlicki et al.,
1994a, 1994b). The accumulation of nascent polypeptides appears to be
related to a failure in one or more chaperone-mediated reactions that
are required for the termination and release reactions of protein
synthesis. The full-length ribosome-bound polypeptides are
enzymatically inactive but are in a partially folded state. They can be
released from the ribosomes as fully active rhodanese by incubation
with the five E. coli chaperones (DnaJ, DnaK, GrpE, GroEL, and
GroES) simultaneously (Kudlicki et al., 1994b). The activation
and release process is blocked by sparsomycin at concentrations that
inhibit the ribosomal peptidyl transferase reaction (Cundliffe, 1980).
Using fluorescence techniques, we demonstrated that incubation of
the ribosomes with DnaJ plus DnaK has an effect on the emission
spectrum of the ribosome-bound nascent CPM-rhodanese that appears to
reflect a DnaJ-dependent reaction in a step of folding, termination, or
release of rhodanese from the ribosome (Kudlicki et al.,
1994c). For these experiments, synthesis of rhodanese was initiated
with CPM-SAc-[
In this report we
present data indicating that the N-terminal amino acid sequence of
rhodanese is critically important for the ribosome-associated,
DnaJ-dependent reaction that is required for correct folding of the
nascent protein into its native form. A deletion mutant lacking the 23
N-terminal amino acids does not form the DnaJ-dependent intermediate
and is not folded into an enzymatically active conformation. This lack
of enzymatic activity was not anticipated from the crystal structure
(Ploegman et al., 1978). A chemically synthesized peptide
corresponding to the 17-amino acid N-terminal sequence of rhodanese
blocks the formation of the DnaJ-dependent intermediate of the
wild-type protein that was detected by fluorescence and prevents the
formation of enzymatically active rhodanese. The relation of the
N-terminal peptide to inhibitory leader peptides studied by Lovett and
co-workers (Gu et al., 1994a, 1994b) is discussed.
The in vitro system for coupled transcription/translation
(usually a 30-µl reaction mixture) has been detailed (Kudlicki
et al., 1992, 1994b). [
In some experiments when
release of ribosome-bound rhodanese was studied, the reaction mixture
after incubation with chaperones or other additions was centrifuged in
an airfuge (Beckman) at 150,000
Fluorescence measurements were carried out on a model
8000C photon-counting spectrofluorometer from SLM-Aminco Instruments
Inc. (Urbana, IL). Spectra were measured at 1-nm intervals at a
scanning rate of 0.5 s/wavelength increment. Unless otherwise
indicated, excitation was at 390 nm. Spectra and relative quantum
yields were normalized on the basis of radioactivity from
[
Precipitation of protein by trichloroacetic acid to
quantitate the amount of rhodanese polypeptides, analysis by
SDS-polyacrylamide gel electrophoresis, and autoradiography were
carried out as before (Kudlicki et al., 1994a). Rhodanese
activity was assayed according to Sörbo (1953) as given in
Tsalkova et al. (1993) and Kudlicki et al. (1994b).
A peptide of 17 amino acids corresponding to the N-terminal sequence
of bovine rhodanese was synthesized by manual solid support peptide
synthesis procedures (Stewart and Young, 1984) by sequential addition
of tert-butyloxycarbonyl-protected L-amino acids
(Bachem, Inc., Torrance, CA, or Applied Biosystems, Foster City, CA).
Similarly, a peptide was synthesized corresponding to the proline-rich
interdomain tether region of rhodanese (amino acids 142-156 of
the bovine rhodanese sequence). The peptides were cleaved from the
resins and the protective groups of reactive side chains removed by
treatment with hydrogen fluoride (Immuno-Dynamics, Inc., La Jolla, CA).
The cleaved peptides were purified by reversed phase high performance
liquid chromatography (HPLC) using nonlinear gradients of acetonitrile
(5-85%) with 0.1% (v/v) trifluoroacetic acid (Pierce) as
described previously (Merrill et al., 1992).
The supernatant
fraction of both the wild-type ( A) and the mutant rhodanese
( B) were incubated with anti-coumarin antibodies to give
changes in the fluorescence emission spectra that are evident in
Fig. 2
. These polyclonal antibodies combine directly with
coumarin and cause a large increase in fluorescence quantum yield and a
shift toward the blue in the emission spectrum that appears to be
associated with binding of the coumarin into a hydrophobic environment
within the antibody. An effect very similar to that shown for
enzymatically active wild-type rhodanese is observed with low molecular
weight model compounds containing coumarin such as the corresponding
derivative of cysteine (data not shown). The anti-coumarin antibodies
produce a much larger increase in fluorescence intensity (from 1.00 to
1.70) with the wild-type protein (Fig. 2, insets) than
with the mutant rhodanese (from 1.11 to 1.30). These differences in
quantum yield of the two proteins are associated with significant
differences in fluorescence anisotropy, increases from 0.18 to 0.37 for
wild-type but from 0.18 to 0.33 for the mutant protein. Our tentative
interpretation of these data is that in contrast to the wild-type
protein only part of the N-terminal coumarin of the mutant is
accessible to the antibodies.
The changes in
fluorescence, especially changes in anisotropy and quantum yield that
are caused by the N-terminal peptide indicate that it affects the
mobility and local environment of the N-terminal coumarin probe of
nascent wild-type rhodanese while the latter is bound as peptidyl-tRNA
to the ribosomes. Although there is a possibility that such effects
might be indirect through the ribosome or mediated by a small amount of
DnaJ that remains associated with the ribosomes throughout their
isolation and subsequent experimental manipulation, our tentative
interpretation is that the synthetic N-terminal peptide interacts
directly with the ribosome-bound wild-type rhodanese chains. It is
somewhat surprising that it does not appear to interact with nascent
peptides of the N-terminal deletion mutant. This may reflect a
requirement for the N-terminal sequence in early steps of folding that
precede the state that is required for the DnaJ/DnaK reaction.
The results presented above indicate that the N-terminal
segment of nascent rhodanese bound to ribosomes as peptidyl-tRNA is
essential for the reaction promoted by DnaJ/DnaK and for folding of the
protein into an enzymatically active conformation. Synthesis of
enzymatically active wild-type rhodanese in the cell-free
transcription/translation system characteristically declines to a very
low rate after about 20 min of incubation with an accumulation on the
ribosomes of full-length rhodanese chains as peptidyl-tRNA. This
nascent protein is enzymatically inactive but can be released as fully
active rhodanese by subsequent incubation of the isolated ribosomes
with the five bacterial chaperones. No similar effects were seen when a
deletion mutant lacking the N-terminal 23 amino acids of wild-type
rhodanese was translated in the cell-free system. Efficient synthesis
of the mutant protein continued at a relatively high rate for 50 min,
and there was less accumulation of full-length rhodanese on the
ribosomes compared with that observed with the wild-type enzyme.
However, the mutant protein that was released from the ribosomes was
enzymatically inactive. This was unanticipated in that examination of
the crystal structure of the wild-type enzyme indicated that the
N-terminal 23-amino acid segment is positioned on the surface of one of
two globular domains.
A chemically synthesized peptide corresponding
to the N-terminal 17 amino acids of the wild-type enzyme efficiently
inhibited synthesis of the wild-type enzyme but had much less effect on
the synthesis of the mutant protein. This peptide blocked the
chaperone-mediated release of the wild-type enzyme from the ribosomes
and prevented formation of the intermediate species that is formed in
the presence of DnaJ/DnaK. A 15-amino acid peptide corresponding to an
internal segment that spans the region between the two globular domains
of the native enzyme had little or no effect comparable with those
observed with the N-terminal peptide, indicating specificity for a
specific amino acid sequence.
The recent report of Mendoza and
Horowitz (1994) may provide insight into the molecular mechanisms that
underlie these complex results. They found that a peptide corresponding
to the N-terminal 23 amino acids of wild-type rhodanese inhibited
refolding of urea-unfolded rhodanese both in the presence and absence
of chaperonin 60 (GroEL). However, the peptide did not block the
interaction of the partially refolded protein with the chaperone. A
13-amino acid peptide corresponding to N-terminal sequence 11-23
of the native enzyme had no comparable effects. We believe these
observations are of special importance for interpretation of the
results presented here because they demonstrate conclusively that the
N-terminal peptide can react directly with a folding intermediate of
rhodanese that is formed in the absence of chaperones or ribosomes and
that this apparently specific interaction can block subsequent folding
of the protein into the native conformation. We suggest that the
phenomena reported here reflect a similar specific interaction of the
N-terminal peptide with a partially folded state of nascent rhodanese
that is bound to the ribosomes as peptidyl-tRNA in addition to possible
interactions of the peptide with either DnaJ/DnaK or the ribosomes.
That the N-terminal peptide has a direct effect on the local
environment of the N-terminal probe on nascent rhodanese is evident
from the spectra of Fig. 5 A. Binding of the peptide
appears to block the subsequent reaction that is mediated by DnaJ/DnaK.
However, the peptide has a much larger effect on fluorescence from the
N-terminal probe if it is added subsequent to the DnaJ/DnaK reaction
(Fig. 5 B). These effects appear to indicate that the
DnaJ/DnaK reaction facilitates binding of the N-terminal peptide in the
immediate vicinity of the N-terminal probe on the nascent protein. This
may be at or near the binding site on the globular domain to which the
N-terminal segment of the protein may be bound as indicated in the
crystal structure.
Considered together we suggest that the results
presented here and in a previous publication (Kudlicki et al.,
1994c) indicate that the N-terminal segment of wild-type rhodanese
facilitates early steps in the correct folding of the nascent protein
within the ribosomes as it is extended vectorially from its N terminus.
Without the N-terminal segment the truncated mutant protein does not
properly undergo these early folding reactions and is released,
apparently through the standard termination reactions. The reaction
that is mediated by DnaJ/DnaK, which is required for wild-type
rhodanese to fold into the native conformation, is dependent upon the
correct form of the partially folded species. The primary effect of the
DnaJ/DnaK reaction may be to position the nascent protein on the
ribosome so that it can undergo subsequent folding reactions such as
those mediated by GroEL/GroES. These chaperones are critically required
in the same reaction mixture with DnaJ/DnaK to form enzymatically
active protein, but it is not known whether or not the reactions they
promote take place while the nascent protein is associated with the
ribosome.
Lovett and his co-workers (Gu et al., 1994a,
1994b) have described a very provocative series of studies involving
the effect of peptides corresponding to the N-terminal sequences of
certain chloramphenicol-inducible genes. These peptides inhibited the
ribosomal peptidyl transferase reaction. Inhibitory peptides appeared
to bind directly to the ribosomes in that their binding was competitive
with erythromycin. Both erythromycin and the peptide footprinted to
overlapping sites in the central loop of domain V of 23 S rRNA (Gu
et al., 1994a). Recently, data were reported indicating that
the inhibitory peptides can block the release factor-dependent
hydrolysis of ribosome-bound Met-tRNA
Fluorescence parameters for wild-type rhodanese and deletion
mutant rhodanese were determined after their synthesis by coupled
transcription/translation with
CPM-SAc-[
An aliquot of the ribosomal fraction (50
pmol of [
Ribosomes containing 4 pmol of
CPM-[
We thank Ronda Barnett for preparing the typescript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helix and that the N terminus is positioned
on the surface of one of the two globular domains (Ploegman et
al., 1978; Hol et al., 1983).
S]Met-tRNA
(
)
to incorporate coumarin at the N terminus of nascent
rhodanese. The fluorescent label does not affect enzymatic activity of
the in vitro synthesized rhodanese that is released from the
ribosome in the presence of the five chaperones.
Materials
Nucleoside triphosphates and E.
coli tRNA were purchased from Boehringer Mannheim;
3-(4-maleimidophenyl)-7-diethylamino-4-methylcoumarin (CPM) was from
Molecular Probes Inc. (Eugene, OR). tRNA,
rifampicin, sparsomycin, and all other biochemicals were from Sigma. A
mixture of ribonuclease A and TI (``RNase Mixture'') was from
Ambion (Austin, TX). [
C]Leucine and
[
S]methionine were purchased from DuPont NEN.
The chaperones DnaK, DnaJ, GrpE, GroEL, and GroES were bought from
Epicentre Technologies (Madison, WI). The plasmids used were pSP65
containing the rhodanese coding sequence under the SP6 promoter
(Kudlicki et al., 1994b) and pET 11d containing the mutant
rhodanese lacking amino acids 1-23 under the T7
promoter.
(
)
Methods
Propagation of the plasmids,
isolation of SP6 RNA polymerase, and preparation of the E. coli cell-free extract (S30) as well as the isolation of the ribosome
fraction from the S30 were carried out as described previously
(Kudlicki et al., 1992). T7 RNA polymerase was isolated by a
procedure identical to the one used for preparation of SP6 RNA
polymerase. E. coli BL21 (pAR1219) cells were used as a source
for T7 RNA polymerase. The preparation of
CPM-SAc-[S]Met-tRNA
was as described
previously (Kudlicki et al., 1994c). Anti-CPM antibodies were
raised in rabbits; the IgG fraction from the serum of the immunized
rabbit was prepared as described (Picking et al., 1992).
C]Leucine was
the radioactive precursor. When
CPM-SAc-[
S]Met-tRNA
was used,
folinic acid was omitted and [
C]leucine was
replaced by unlabeled leucine. For some of the experiments described
below, reaction mixtures were enlarged to 0.9 ml. They contained salts
and low molecular weight components (Kudlicki et al., 1994b)
including 5 mM Na
S
O
. About
60 A
units of unwashed ribosomes were incubated
with 10-15 µg of nonlinearized plasmid and the appropriate
RNA polymerase. After incubation for 30 min at 37 °C, the sample
was loaded over 0.6 ml of 0.5 M sucrose in 20 mM
Tris-HCl, pH 7.5, 10 mM Mg(OAc)
, 30 mM
NH
OAc, 1 mM dithiothreitol (solution A) and
centrifuged for 45 min at 45,000 rpm (Ti-50 rotor, Beckman). The
resulting supernatant was collected and saved. The sucrose layer was
removed, and the ribosomal pellet was rinsed and then resuspended in 60
µl of solution A. Immediately after centrifugation, the supernatant
fraction (about 0.9 ml) was treated with ribonucleases A and T1 (0.1 mg
and 2,000 units/ml, respectively) for 15 min at 37 °C to degrade
the remaining CPM-SAc-[
S]Met-tRNA
.
The resulting reaction mixture was then chromatographed on a Sephadex
G-100 column (1
20 cm) equilibrated in solution A to separate
newly synthesized CPM-labeled rhodanese from low molecular weight
degradation products containing coumarin and
[
S]methionine.
g for 40 min. Then the
supernatant was pipetted off, and the ribosomal pellet was resuspended
in solution A.
S]methionine that was present in the sample as
CPM-SAc-[
S]methionine incorporated into protein.
Relative fluorescence quantum yields were determined from integrated
emission spectra. The relative quantum yield of CPM-rhodanese in the
supernatant fraction was taken as 1.00, and all other quantum yields
are given relative to this value. Fluorescence anisotropy was
determined as described in Odom et al. (1984) at an emission
wavelength of 480 nm. For the fluorescence experiments, an aliquot of
the resuspended ribosomes or of the treated supernatant fraction (see
above) was incubated in the cuvette in a total volume of 420 µl
containing salts and low molecular weight components as above for the
coupled transcription/translation but without amino acids and without
UTP and CTP (solution B). After the spectrum was taken, the components
to be tested were added in a minimum volume. Additions were as follows:
anti-CPM IgG, 0.1 mg; and chaperones GroES, 1.6 µg; GroEL, 6
µg; DnaK, 4 µg; DnaJ, 2 µg; GrpE, 3 µg. The cuvette was
incubated for 5 min at 20 °C before the emission spectrum was taken
again.
Cell-free Synthesis of Wild-type Rhodanese and Its
N-terminal Deletion Mutant
A deletion mutant of the bovine
rhodanese gene was constructed that lacked codons for the N-terminal 23
amino acids of the wild-type enzyme. The coding sequence of the mutant
gene started with ATG followed by the GTG codon for valine 24. Protein
was synthesized in the cell-free E. coli transcription/translation system from plasmids containing either
the mutant or wild-type gene. Mutant rhodanese was synthesized more
efficiently than wild-type protein in this system (Fig. 1). At
the indicated times, aliquots were centrifuged, and then the resulting
supernatant fraction and the resuspended ribosomes were analyzed
separately. The initial rate of leucine incorporation into ribosome and
soluble fraction was very similar for the mutant and wild-type
proteins. But marked differences became apparent during the later
stages of the incubation. The mutant protein that was bound to the
ribosomes reached a maximum of about 23 pmol after 10 min of incubation
(Fig. 1 A). This value remained essentially constant
during the next 40 min of incubation, whereas total synthesis continued
at a relatively high rate throughout the 50-min incubation period. In
contrast, the newly formed wild-type protein accumulated on the
ribosomes at a nearly linear rate for 20 min to reach a maximum of near
50 pmol (Fig. 1 B). The rate of total synthesis and
release of protein into the soluble fraction declined to near zero
after about 30 min of incubation. The enzyme that was released appeared
to be folded into the native conformation as judged by its specific
enzymatic activity. Neither the supernatant nor the ribosome-bound
forms of the mutant protein were enzymatically active, whereas the
specific enzymatic activity of the wild-type protein in the supernatant
fraction was near that of the native enzyme. The wild-type rhodanese
that was bound to the ribosomes was enzymatically inactive as
previously reported (Kudlicki et al., 1994a, 1994b).
Figure 1:
Synthesis of wild-type
rhodanese and its 1-23 N-terminal deletion mutant in the
cell-free transcription/translation system. The deletion mutant
( A) and wild-type ( B) rhodanese were synthesized
in vitro in the presence of [C]leucine
(40 Ci/mol). The volume of each reaction mixture was 240 µl, and a
35-µl aliquot was taken for each time point. The ribosomes and
supernatants were separated by centrifugation after the time shown on
the abscissa. A 15-µl aliquot of the supernatant
( A, opentriangles; B, opensquares) and the resuspended ribosome fraction
( A, filledtriangles; B, filledsquares), respectively, was taken to determine the amount
of [
C]leucine incorporated into protein, and an
equal aliquot was used to measure rhodanese enzymatic activity.
Opencircles and filledcircles represent rhodanese specific activity for supernatant and ribosome
fractions, respectively.
Analysis by SDS-polyacrylamide gel electrophoresis and
autoradiography indicated that nearly all of the mutant or wild-type
polypeptides released into the supernatant fraction throughout the
incubation were of the size anticipated for the full-length product
(results not shown). About half of the ribosome-bound peptides
consisted of full-length rhodanese chains (not shown). In the case of
wild-type rhodanese, these were more than twice the amount compared
with the ribosome-bound mutant rhodanese and constituted a high portion
of the total protein synthesized. These results are consistent with the
hypothesis that the wild-type protein is processed through a
ribosome-dependent step in folding that limits its termination and
release from the ribosome, whereas termination and release of the
mutant protein is not subjected to this limiting step.
Fluorescence Analyses of the Deletion Mutant and
Wild-type Rhodanese
Fluorescence techniques were used to
investigate the differences indicated above in the release and folding
of the N-terminal deletion mutant and wild-type rhodanese. To this end,
both proteins were synthesized with
CPM-SAc-[S]Met-tRNA
to incorporate
coumarin at their N termini. After 30 min of incubation, the reaction
mixtures were centrifuged to separate the supernatant from the
ribosomes. After ribonuclease treatment and gel permeation
chromatography of the supernatant fraction to remove unincorporated
CPM-SAc-[
S]methionine (see ``Experimental
Procedures''), both the supernatant fraction and the resuspended
ribosomes were analyzed by fluorescence. The results are summarized in
. As described previously, fluorescence intensities were
normalized on the basis of [
S]methionine
incorporated into protein (Kudlicki et al., 1994c), and the
quantum yield of wild-type enzymatically active rhodanese in the
supernatant was set at 1.00. The quantum yield is higher (1.20
versus 1.00) for the enzymatically inactive mutant rhodanese
in the supernatant fraction. A higher quantum yield was observed
previously for wild-type rhodanese released from the ribosome by
puromycin or by DnaK only (Kudlicki et al., 1994c). Both of
these forms of wild-type rhodanese are enzymatically inactive. The
results appear to indicate that the N-terminal conformations of the
enzymatically active wild-type protein and of the wild-type protein
released by puromycin or DnaK are different as is the environment of
the N-terminal coumarin of the released mutant protein. The
ribosome-bound forms of both the wild-type and mutant protein have
lower quantum yields than their soluble counterparts as shown in
. The maxima of the emission spectra of both the
supernatant and ribosome-bound forms of the mutant protein are
significantly higher than the corresponding values for the wild-type
protein. Fluorescence anisotropy is relatively high for the
ribosome-bound forms of both proteins, as expected.
Figure 2:
Fluorescence emission spectra of CPM
wild-type and mutant rhodanese before and after incubation with
anti-coumarin antibodies. Wild-type rhodanese and mutant rhodanese were
synthesized in the presence of
CPM-SAc-[S]Met-tRNA
(4,000 Ci/mol),
and then the reaction mixtures were centrifuged. The resulting
supernatant fractions were treated with ribonuclease and
chromatographed over Sephadex G-100, and then the emission spectra were
taken in the absence of antibodies and after incubation for 5 min at
room temperature with anti-coumarin IgG. A, wild-type
rhodanese; B,
1-23 mutant rhodanese.
Insets, quantitative fluorescence data determined in the
absence ( -IgG) and in the presence ( +IgG)
of anti-coumarin antibodies. RelQ, relative
fluorescence quantum yield (as explained in Table I); A,
fluorescence anisotropy;
, wavelength of emission
maximum.
Fluorescence emission spectra of the
ribosome-bound rhodanese in the presence and absence of anti-coumarin
antibodies were also taken. In the absence of the antibodies the
wild-type and the deletion mutant gave generally similar spectra
(Fig. 3, A, B, -IgG), the
emission maximum of the mutant being at a somewhat longer wavelength
than that of the wild-type (483 versus 471 nm, ).
Addition of DnaJ plus DnaK gave only minor spectral changes (shown in
Fig. 5
). However, a pronounced difference between the spectra of
the ribosome-bound wild-type and mutant protein was observed in the
presence of antibodies after the ribosomes had been incubated with DnaJ
plus DnaK. The ribosome-bound wild-type protein exhibited a new
fluorescent species with an emission maximum near 435 nm
(Fig. 3 A). This is the intermediate species that
apparently is produced by a reaction promoted by DnaJ as previously
reported (Kudlicki et al., 1994c). This fluorescent species
was not detected with the ribosome-bound mutant protein
(Fig. 3 B). The spectrum of the mutant protein that was
obtained in the presence of antibodies was very similar to the
corresponding spectra exhibited by both the wild-type and mutant
protein that had been released into the soluble phase ( cf.
Fig. 2
). We interpret these results to indicate that the
ribosome-bound mutant rhodanese is unreactive in the reactions promoted
by DnaJ plus DnaK and that the N-terminal sequence of rhodanese is
critically important for formation of the DnaJ- plus DnaK-dependent
intermediate. This intermediate may be a prerequisite for correct
folding into enzymatically active enzyme that is promoted in the
presence of the other chaperones.
Figure 3:
Mutant and wild-type ribosome-bound
nascent rhodanese after incubation of the ribosomes with DnaJ plus DnaK
and then with anti-CPM antibodies. Wild-type and N-terminal deletion
mutants of rhodanese were synthesized from
CPM-SAc-[S]Met-tRNA
(4,000 Ci/mol)
by coupled transcription/translation, and then ribosomes with bound
rhodanese polypeptides were separated from the reaction mixture by
centrifugation. The resuspended ribosomes were incubated with DnaJ plus
DnaK and ATP for 5 min at 25 °C. The fluorescence emission spectra
were taken in the absence of antibodies and after incubation with
anti-coumarin IgG. A, wild-type rhodanese; B,
1-23 mutant rhodanese.
Figure 5:
The effects of the N-terminal peptide and
DnaJ/DnaK on the emission spectrum of ribosome-bound wild-type
CPM-rhodanese in the presence and absence of anti-coumarin IgG.
Ribosomes bearing wild-type CPM-rhodanese nascent peptides were
incubated with the N-terminal peptide, DnaJ/DnaK, and anti-coumarin IgG
as described in Table III. A, incubation first with the
N-terminal peptide. Spectrum1, after incubation
without additions; spectrum2, after incubation with
N-terminal peptide; spectrum3, as in spectrum2 and then incubation with DnaJ/DnaK; spectrum4, as in spectrum3 and then incubation
with anti-coumarin IgG. B, incubation first with DnaJ/DnaK.
Spectrum1, after incubation without additions;
spectrum2, after incubation with DnaJ/DnaK;
spectrum3, as in spectrum2 and
then then incubation with N-terminal peptide; spectrum4, as in spectrum3 and then incubation
with IgG.
Effects of the N-terminal Rhodanese Peptide on Cell-free
Synthesis of Rhodanese
The results with the N-terminal deletion
mutant that were presented in the preceding sections prompted
considerations of the effects that the N-terminal peptide itself might
have on the in vitro synthesis and activation of mutant and
wild-type rhodanese. Two peptides were chemically synthesized and
purified by HPLC. The synthetic peptide used in the experiments
described below corresponded to amino acids 1-17 of the wild-type
enzyme. As a control a second peptide was synthesized that corresponded
to an internal 15-amino acid sequence, amino acids 142-156 that
connect the N-terminal and C-terminal globular domains of the wild-type
enzyme in its native conformation (Ploegman et al., 1978).
This is called the ``tether peptide'' in the description
given below. Neither of these peptides had any effect on the
fluorescence or enzymatic activity of wild-type rhodanese that had been
released from the ribosomes or the corresponding mutant protein that is
enzymatically inactive (data not shown). Interesting differences in the
activity of the two peptides were observed when they were added
separately to reaction mixtures in which either the wild-type or the
deletion mutant of rhodanese was synthesized (Fig. 4). The
N-terminal peptide caused considerably higher inhibition of synthesis
of the wild-type protein than the deletion mutant. The tether peptide
had no effect on the coupled transcription/translation of either the
wild-type or the deletion mutant rhodanese coding sequence.
Figure 4:
Effect of rhodanese N-terminal peptide on
rhodanese synthesis during coupled transcription/translation. Either
the N-terminal peptide ( squares) or the tether peptide
( triangles) was added in the indicated amount to the coupled
transcription/translation reaction mixtures (30 µl total volume)
containing either wild-type ( filledsymbols) or
deletion mutant ( opensymbols) rhodanese coding
sequence. The amount of protein synthesized in the whole reaction
mixture (30 µl) was analyzed by incorporation of
[C]leucine as described under
``Methods.'' The 100% values were 94 pmol of
[
C]leucine incorporated into wild-type rhodanese
and 121 pmol of [
C]leucine incorporated into
mutant protein.
Previously we demonstrated that full-length rhodanese polypeptides
accumulate on the ribosomes as peptidyl-tRNA and that this nascent
rhodanese can be released in an enzymatically active form during an
incubation of the ribosomes with all five chaperones under reaction
conditions in which the formation of additional peptide bonds could not
be detected (Kudlicki et al., 1994b). The data given in
indicate that the N-terminal peptide inhibits this release
and activation of ribosome-bound wild-type rhodanese. After coupled
transcription/translation, the ribosomes bearing nascent rhodanese were
isolated by centrifugation and then incubated with the additions shown
in the table. After this incubation the ribosomes were reisolated by
centrifugation. The resulting supernatant was analyzed for amount of
protein released and enzymatic activity. These results are given in
. Without the N-terminal or tether peptide, the chaperones
(DnaJ, DnaK, GrpE, GroEL, and GroES) caused the release of more than
half of the polypeptides from the ribosomes as determined by
incorporated [C]leucine. Nearly all of the
chaperone-released protein is full-length rhodanese; shorter peptides
remain bound to the ribosomes (Kudlicki et al., 1994b). The
protein that is released from the ribosomes appears to be in the native
conformation as inferred from its specific enzymatic activity, which
approaches the value expected for native rhodanese (Jarabak and
Westley, 1974). When the N-terminal peptide was included in the
reaction mixture with the chaperones, release of rhodanese from the
ribosomes was reduced from 27 to 8 pmol of leucine (). The
small amount of protein that was released had the specific enzymatic
activity of the native enzyme. The tether peptide had no apparent
effect on release or the specific enzymatic activity of the released
wild-type protein.
Effect of the N-terminal Peptide on Fluorescence from
Ribosome-bound CPM-rhodanese
The effect of the N-terminal
peptide on the DnaJ/DnaK-dependent emission spectrum of ribosome-bound
wild-type CPM-rhodanese that is seen in the absence or presence of
anti-coumarin antibodies was examined, and the results are shown in
Fig. 5
and I. The most striking effect is that the
emission peak with a maximum at 435 nm is not observed if the ribosomes
bearing CPM-rhodanese are incubated with the N-terminal peptide before
they are incubated with DnaK/DnaJ (Fig. 5 A) and then
with anti-coumarin IgG ( curve4). The maximum of the
emission spectrum and increase in quantum yield (I) that
occur with the addition of the antibodies are very similar to those
seen with the N-terminal deletion mutant (Fig. 2 B). It
should be noted from Fig. 5 A and I that the
N-terminal peptide itself without DnaJ/DnaK or antibodies causes a
significant increase in the quantum yield and a pronounced drop in
fluorescence anisotropy, from 0.89 to 0.97 and 0.306 to 0.235,
respectively (I). Subsequent incubation with DnaJ/DnaK
causes an increase in anisotropy to 0.266 and an increase in quantum
yield to 1.03. In contrast, if the CPM-rhodanese ribosomes are
incubated with DnaJ/DnaK before the incubation with the N-terminal
peptide, the emission peak with a maximum at 435 nm is observed after
the addition of the antibodies, and the maximum of the largest peak in
the emission spectrum is at about 473 nm (Fig. 5 B).
Incubation of the ribosomes with DnaJ/DnaK only had little direct
effect on fluorescence (Fig. 5 B, lines 1 and
2), but the subsequent incubation with the N-terminal peptide
causes a relatively large increase in quantum yield
(Fig. 5 B, line3; I C,
quantum yield of 0.92 versus 1.29). Note that the subsequent
incubation with anti-coumarin IgG after incubation with DnaJ/DnaK and
then the N-terminal peptide causes a much smaller increase in the
fluorescence intensity at 473 nm (Fig. 5 B, line4) than was observed in the absence of the N-terminal
peptide but in an otherwise similar experiment
(Fig. 3 A). The tether peptide had no detectable effects
similar to those described for the N-terminal peptide (data not shown).
Also, the N-terminal peptide had no effect on fluorescence from
ribosome-bound mutant CPM-rhodanese comparable with those shown above
for wild-type rhodanese (data not shown).
or reaction of this
tRNA species with puromycin (Moffat et al., 1994). These
results clearly indicate that the binding site for the inhibitory
peptide is on the ribosome. The observations have intriguing
similarities to the results reported here. However, as yet we have been
unable to show that the 17-amino acid N-terminal rhodanese peptide at
concentrations similar to those used in the experiments described above
has a direct inhibitory effect on the peptidyl transferase reaction.
Despite this failure to detect direct inhibition of the peptidyl
transferase reaction, our results indicate that some step of the normal
release mechanism fails during the translation of wild-type rhodanese
in the cell-free system. We suggest that the observations reported by
Lovett and his co-workers (Gu et al. 1994a, 1994b) and those
reported here reflect different aspects of the same steps in the
sequence of reactions by which nascent proteins are folded on the
ribosomes and released by the termination reactions. We also suggest
that the nascent wild-type rhodanese folds into a conformation in which
its N terminus is brought into a position within the ribosome in which
it can interact with a peptide-binding site in the 23 S rRNA that was
observed by Lovett and his co-workers.
Table: Fluorescence characteristics of
CPM-SAc-Met-rhodanese (free in solution and bound to ribosomes)
S]Met-tRNA
as the initiator
tRNA. After a 30-min incubation at 37 °C, the reaction mixtures
were centrifuged to obtain the ribosomal fraction and supernatant and
analyzed as described under ``Methods.''
Table: The effects of the N-terminal peptide on
the chaperone-dependent activation and release of ribosome-bound
wild-type rhodanese
C]leucine in protein) isolated after
coupled transcription/translation (see legend of Table I and
``Methods'') was incubated in the absence or presence of the
N-terminal peptide (4 µg), tether peptide (5 µg), chaperones
(amounts: 3.5 µg of GroEL, 0.8 µg of GroES, 2 µg of DnaK, 1
µg of DnaJ, 1 µg of GrpE), plus low molecular weight components
as specified under ``Experimental Procedures.'' The volume of
reaction mixture was 30 µl. After the 30-min incubation, the
ribosomes were separated from the supernatant by a second
centrifugation. The resulting supernatant fractions were analyzed for
the amount of protein that had been released from the ribosomes and its
enzymatic activity.
Table: Effect of the N-terminal peptide on
DnaJ/DnaK-mediated changes in fluorescence from wild-type CPM-rhodanese
bound to ribosomes
S]methionine in nascent rhodanese peptides
were incubated for 5 min at room temperature under nontranslating
conditions with the indicated additions. For B and C the ribosomes were
incubated first with either the N-terminal peptide or with DnaJ/DnaK,
as indicated, and then the missing components, either the N-terminal
peptide or DnaJ/DnaK, were added, and the incubation continued for an
additional 5 min. Where indicated, anti-coumarin antibodies were then
added, and the incubation continued for an additional 5 min. The
fluorescence characteristics of the reaction mixtures were determined
as described for Table I. Amounts of antibodies, DnaJ, DnaK, and
N-terminal peptide added were 0.1 mg, 2.5 µg, 5 µg, and 4
µg, respectively. Abbreviations are as defined in Table I.
S]SAc-Met-tRNA
, initiator tRNA
that has been aminoacylated with methionine and then labeled on the
methionine amino group via mercaptoacetic acid by CPM; CPM,
3-(4-maleimidophenyl)-4-methyl-7-(diethylamino)coumarin; HPLC, high
performance liquid chromatography; CPM-rhodanese, rhodanese labeled at
its N-terminal methionine with CPM; anti-CPM IgG, anti-coumarin
polyclonal antibodies produced in rabbits.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.