J Biol Chem, Vol. 275, Issue 3, 1781-1786, January 21, 2000
Fluorophores at the N Terminus of Nascent Chloramphenicol
Acetyltransferase Peptides Affect Translation and Movement through
the Ribosome*
Vasanthi
Ramachandiran,
Charles
Willms,
Gisela
Kramer, and
Boyd
Hardesty
From the Department of Chemistry and Biochemistry, University of
Texas, Austin, Texas 78712
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ABSTRACT |
Structurally different fluorescent probes
were covalently attached to methionyl-tRNAf and
tested for their incorporation into nascent peptides and full-length
protein using an Escherichia coli cell-free coupled
transcription/translation system. Bovine rhodanese and bacterial
chloramphenicol acetyltransferase (CAT) were synthesized using
derivatives of cascade yellow, eosin, pyrene, or coumarin attached to
[35S]Met-tRNAf. All of the probes tested were
incorporated into polypeptides, although less efficiently when compared
with formyl-methionine. Eosin, the largest of the fluorophores used
with estimated dimensions of 20 × 11 Å, caused the largest
reduction in product formed. The rate of initiation was reduced with
the fluorophore-Met-tRNAf compared with
fMet-tRNAf with pyrene having the least and eosin the
biggest effect. Analysis of the nascent polypeptides showed that the
modifications at the N terminus affected the rate at which nascent CAT
peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S
ribosomal subunit. Fluorescence measurements indicate that the probe at
the N terminus of nascent pyrene-CAT peptides is in a relatively
hydrophilic environment. This finding is in agreement with recent data
showing cross-linking of the N terminus of nascent peptides to
nucleotides of the 23 S ribosomal RNA.
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INTRODUCTION |
Initiation of protein synthesis by a specific initiator
tRNAMet at an AUG codon is a universally conserved step in
gene expression for both eukaryotes and prokaryotes. In prokaryotes,
methionine linked to the initiator tRNAMet is formylated to
give fMet-tRNAf.1
However, peptide initiation can be carried out with other
N-acyl derivatives of Met-tRNAf, many of which
can be synthesized chemically from Met-tRNAf.
We have synthesized coumarin
maleimide-S-acetyl-Met-tRNAf to incorporate a
fluorescent probe at the N terminus of polypeptides during their
synthesis in a cell-free coupled transcription/translation system
derived from Escherichia coli (1-4). A considerable amount of information on the role of the molecular chaperones DnaJ and DnaK in
folding of the nascent protein and on cotranslational folding of
nascent peptides was obtained through this method. In addition, we
showed recently (5) that incorporation of coumarin at the N terminus of
bacterial chloramphenicol acetyltransferase (CAT) resulted in increased
ribosomal pausing at the already existing pause sites provided
translation was slowed. Translational pause sites and their potential
causes have been extensively discussed in this publication.
The studies cited above have prompted the question of what are the
limits in terms of size and chemical character of the modification on
the N terminus of nascent peptides under optimal translation conditions. Will the ribosomes initiate with bulky (pyrene, eosin) or a
charged group (cascade yellow) covalently attached to the 
-amino
group of methionine on the initiator-tRNA as efficiently as with
fMet-tRNAf? Initiation of protein synthesis is usually the
rate-controlling step and a major point for regulation of translation
(6). Do the fluorophores at the N terminus affect the amount and the
rate at which polypeptides are formed after they are initiated?
Are they incorporated into native full-length protein? The last two
questions imply that the bulky modification at the N terminus may
hinder the required folding of the nascent peptide to first pass
through the tunnel inside the large ribosomal subunit (7), then to
acquire the three-dimensional structure of the native protein.
Here we report the effect on translation of three additional N-terminal
fluorophores (pyrene, cascade yellow, eosin), which vary in size and
structure compared with coumarin. Synthesis of bovine rhodanese (RHO, a
thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) or bacterial CAT (EC
2.3.1.28) was initiated in the presence of
fluorophore-Met-tRNAf. Formation of nascent peptides and
full-length product was analyzed and compared with polypeptides formed
with N-formyl-methionine at the N terminus. The results indicate that
RHO and CAT could be produced when pyrene-Met or cascade yellow-Met
were at the N terminus of the protein; however, using
eosin-Met-tRNAf, the translational machinery worked
less efficiently. With all fluorophore-Met-tRNAf species,
peptide initiation was an impaired step. With CAT, but not with RHO,
pausing during translation was increased, resulting in accumulation of
low molecular weight nascent peptides. The results are discussed under
the aspect of folding of the nascent polypeptides on the ribosomes.
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EXPERIMENTAL PROCEDURES |
Materials
3-(4-Maleimidophenyl)-7-(diethylamino-4-methyl) coumarin,
N-(1-pyrene)-maleimide, eosin-5-maleimide, and cascade
yellow succinimidyl ester were from Molecular Probes, Inc. (Eugene,
OR). The structures of coumarin-maleimide,
N-(1-pyrene)-maleimide, and eosin-5-maleimide are given in
Ref. 25. The structure of the succinimidyl ester of cascade yellow is
given in Ref. 26.
tRNAfMet, rifampicin, and all other
biochemicals were from Sigma. [35S]Methionine was
purchased from NEN Life Science Products. Puromycin-CPG and
5'-dimethoxytrityl-N-acetylcytidine,
2'-O-TBDMS-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite were purchased from Glen Research (Sterling, VA). Cytidylic
acid-puromycin (C-puro) was synthesized from these components by Dr.
John Lee (Department of Biochemistry, University of Texas Health
Science Center, San Antonio, TX). Low molecular weight protein
standards for SDS-PAGE were from Promega (Madison, WI). The original
rhodanese plasmid was a kind gift from Dr. Paul Horowitz (University of Texas Health Science Center, San Antonio, TX), and the plasmid containing CAT coding sequence was provided by Dr. A. S. Spirin (Institute of Protein Research, Pushchino, Russia). Both the coding sequences of rhodanese and CAT were inserted under the T7 promoter in a
pGEM vector.
Methods
Synthesis of Fluorophore-Met-tRNAf
Species--
Coumarin-SAc-Met-tRNAf was prepared as
described previously (1). Both
eosin-succinimido-SP-Met-tRNAf and
pyrene-succinimido-SP-Met-tRNAf were synthesized by
the same protocol except that
3,3-dithiobis(sulfosuccinimidyl)propionate (DTSSP) was
used instead of succinimide monoester of dithiodiglycolic acid. DTSSP
is water-soluble and will cross-link two tRNA molecules, even though it
is present at a high molar excess of the latter. The product including
any DTSSP-([35S]Met-tRNA)2 that was formed
was reduced by dithiothreitol. The resulting thiopropionate derivative
of [35S]Met-tRNAf was then reacted with
maleimides of eosin or pyrene.
Cascade yellow-[35S]Met-tRNAf was synthesized
by reacting the amino group of [35S]Met-tRNAf
with the succinimidyl ester of cascade yellow followed by ethanol
precipitation. HPLC purification was carried out for all the modified
Met-tRNA species essentially as described for coumarin-Met-tRNAf (1). The elution solvents used for HPLC separation of the Met-tRNAf species were 20 mM
Tris acetate (pH 7.5), 10 mM magnesium acetate, 400 mM NaCl (solvent A), and 60% methanol in solvent A
(solvent B). Coumarin-Met-tRNAf and
eosin-Met-tRNAf were eluted from the C3 column with 50% of
solvent B, whereas pyrene-Met-tRNAf eluted with 80% of
solvent B. Cascade yellow-Met-tRNAf was eluted with 20% of
solvent B.
The Cell-free System--
Propagation of the plasmids, isolation
of T7 RNA polymerase, and preparation of the E. coli
cell-free extract (S30) were carried out as described (8). The in
vitro coupled transcription/translation assay was used (9) with
some modifications in the salt concentration of the reaction mixture,
which included 30 mM of
(NH4)2S2O3. However, unfractionated tRNA and folinic acid were omitted. Bovine RHO and
CAT were synthesized using f[35S]Met-tRNA or
fluorophore-[35S]Met-tRNA (generally at 10,000 Ci/mol) as
the radioactive precursors and the E. coli S30 fraction with
non-linearized plasmids and T7 RNA polymerase plus rifampicin. In the
absence of plasmid, trichloroacetic acid-insoluble material was less
than 5% of the polypeptides formed in the presence of plasmid. These
blank values were subtracted.
Radioactive Labeling of C-puro and Its Reaction with Nascent
Polypeptides on the Ribosomes--
About 40 nmol of C-puro were
incubated for 30 min at 37 °C in a total volume of 500 µl with 150 units of T4 polynucleotide kinase (Life Technologies, Inc.) in
"forward" reaction buffer in the presence of 0.2 mM
[
-32P]ATP, 5000 Ci/mol. Then, the reaction mixture was
held at 65 °C for 10 min to inactivate the enzyme. About 1.5 µl of
this preparation was added to each 15 µl of the
transcription/translation reaction mixture after its incubation for the
indicated time at 37 °C, and then the incubation at 37 °C was
continued for 10 min. About a 7-fold volume of 7% cold trichloroacetic
acid was added and the sample prepared for polyacrylamide gel
electrophoresis, which was carried out as described below.
Gel Electrophoresis and Enzyme Assays--
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Tricine was
performed essentially as described by Schägger and von Jagow
(10). After the gels were dried, they were exposed to a phosphor screen
(35 × 43 cm) and scanned in a PhosphorImager (Molecular Dynamics).
RHO activity was assayed according to the method of Sörbo (11) as
given by Kudlicki et al. (9). CAT enzyme activity was
assayed by the method of Sleigh (12).
Sample Preparation for Fluorescence Measurements--
RHO was
synthesized using cascade yellow- or
pyrene[35S]Met-tRNAf to initiate protein
synthesis. After coupled transcription/translation under standard
conditions, cascade yellow-RHO or pyrene-RHO was purified as described
for coumarin-labeled RHO (1). Fractions containing fluorophore-RHO
after Sephadex G100 chromatography were used for fluorescence
measurements. These were carried out on a model 8000C photon counting
spectrofluorometer from SLM-Aminco (Urbana, IL.). Emission spectra were
recorded at 1 nm intervals using an excitation wavelength of 408 nm for
cascade yellow and 338 nm for pyrene.
In another set of experiments, nascent CAT peptides were synthesized
with pyrene-[35S]Met-tRNAf for 30 min at
37 °C. Then, the ribosomal fraction was isolated by Airfuge
(Beckman) centrifugation and resuspended in 400 µl of 20 mM Tris-HCl, pH 7.5, 10 mM
Mg(OAc)2, 30 mM (NH4OAc), and 1 mM dithiothreitol. The fluorescence emission spectrum was recorded with an excitation wavelength of 338 nm. After the spectrum was taken, 6 µl of [32P]C-puro were added to the
sample, which was then incubated for 10 min at 37 °C before the
emission spectrum was recorded again. Both the spectrum and the
relative quantum yield were normalized on the basis of radioactivity
from [35S]Met. Relative quantum yields were determined
from the integrated emission spectra. Anisotropy was calculated as
described in Ref. 13.
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RESULTS |
Incorporation of Coumarin, Cascade Yellow, Eosin, and Pyrene
Derivatives of Methionine at the N Terminus of
Polypeptides--
Coupled transcription/translation in the in
vitro E. coli system provides a method by which a modified
methionine can be incorporated from derivatized Met-tRNAf
into a nascent peptide. This approach has been used here to study
whether fluorescent probes differing in size and charge can be
incorporated at the N terminus of bovine RHO (33 kDa, Ref. 14) or CAT
(25.6 kDa, Ref. 15) nascent peptides.
First, we analyzed whether the fluorophore-Met modification at the N
termini of RHO or CAT affected the extent to which these polypeptides
were produced in comparison to the initiation with fMet-tRNAf. Production of full-length protein released from
the ribosomes was monitored by their enzymatic activity (cf.
Ref. 9). The data are presented in Table
I. For these experiments, polypeptide
synthesis was carried out in the presence of
f[35S]Met-tRNAf or
fluorophore-[35S]Met-tRNAf. The radioactively
labeled methionine can be incorporated only at the N terminus of the
proteins; the incubation was continued for 30 min to achieve maximal
extension of the polypeptides initiated with the labeled initiator
tRNA. Previous unpublished
data2 showed that
coumarin-Met covalently attached to a peptide cannot serve as a
substrate for deformylase and methionine aminopeptidase. In addition,
earlier unpublished observations2 indicated that the
thioether bond between coumarin and the thiol derivative of methionine
is chemically stable under the incubation conditions used.
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Table I
Synthesis of RHO and CAT initiated with fMet-tRNAf or
fluorophore-Met-tRNAf and enzymatic activity of the product
RHO or CAT was synthesized by coupled transcription/translation under
optimal conditions (using 5 µl of S30/30 µl of reaction mixture)
with CPM-SAc-[35S]Met-tRNAf, cascade
yellow-[35S]Met-tRNAf,
eosin-SP-[35S]Met-tRNAf , or fMet-tRNAf as
the initiator tRNA. After 30 min of incubation at 37 °C, an aliquot
from the 30-µl reaction mixture was precipitated by trichloroacetic
acid and its radioactivity determined. Another aliquot was used to
measure the enzyme activities. The data presented are calculated for a
30-µl reaction mixture. Values for incorporation of the
[35S]methionine derivatives in the absence of added plasmid
were 0.08 ± 0.01 pmol. This value was subtracted. Similarly,
enzymatic activity determined in samples lacking plasmid was subtracted
from the values given for enzyme activity. The data presented are the
average values from three separate experiments.
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The data in Table I indicate a reduction in incorporation of
radioactively labeled N-terminal methionine of 40-60% for both RHO
and CAT when peptide synthesis was initiated with
fluorophore-Met-tRNAf compared with fMet-tRNAf.
In parallel, the measured enzymatic activities were reduced; yet, the
fact that enzymatic activity was associated with the
fluorophore-Met-RHO or -CAT polypeptides that were produced indicates
that apparently full-length protein was formed and folded into its
native structure. Note, however, that CAT activity was reduced more
strongly than would have been expected from incorporation of
radioactivity from Met when synthesis was initiated with
fluorophore-Met-tRNAf.
Earlier observation by Kudlicki et al. (1) indicated that
full-length rhodanese whose synthesis was initiated with
coumarin-Met-tRNAf contained coumarin at its N terminus. We
tested whether pyrene and cascade yellow can be detected at the N
termini of RHO polypeptides released from the ribosomes. Both pyrene
and eosin derivatives of Met-tRNAf were synthesized by the
same reactions and offer to be equally stable. Cascade yellow, on the
other hand, is a zwitterion, has a more flexible structure, and is
linked to the -amino group of methionine by reaction of its
succinimidyl ester.
After coupled transcription/translation in the presence of pyrene- or
cascade yellow-Met-tRNAf, the ribosomal and the supernatant fractions were separated. Released pyrene- or cascade yellow-labeled RHO was purified by gel filtration chromatography (1). Their fluorescence spectra are shown in Fig. 1
in comparison to the respective fluorophore-Met-tRNAf. The
cascade yellow-RHO spectrum (panel A) is shifted
to the blue with a maximum of the emission spectrum of 518 nm compared
with the spectrum of the fluorophore-Met-tRNAf whose
emission maximum is at 542 nm. Pyrene-labeled RHO (panel B) with an emission maximum of 398 nm showed a large
increase in quantum yield compared with pyrene-Met-tRNAf,
indicating that this environmentally sensitive probe is in a more
hydrophobic surrounding at the N terminus of rhodanese. The data
presented in Fig. 1 demonstrate that pyrene and cascade yellow are
indeed present on the protein, which was initiated with the respective fluorophore-Met-tRNAf.
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Fig. 1.
Fluorescence emission spectra of cascade
yellow- and pyrene-labeled rhodanese compared with pyrene- and cascade
yellow-Met-tRNAf. Cascade yellow- or pyrene-labeled
RHO was purified as described under "Experimental
Procedures." Fluorescence spectra were recorded for the pooled column
fractions containing 7.5 pmol of cascade yellow-labeled
(panel A, spectrum 2) or
7.0 pmol of pyrene-labeled (panel B,
spectrum 2) RHO in comparison to 10 pmol of
cascade yellow- or 5 pmol of pyrene-Met-tRNAf
(spectra 1). Excitation wavelengths were 408 and 338 nm for cascade yellow (A) or pyrene
(B), respectively.
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The Rate of Initiation Is Affected When RHO or CAT Syntheses Are
Initiated with Fluorophore-Met-tRNAf Compared with
fMet-tRNAf--
The results shown in Fig.
2 demonstrate that under the conditions
used, the structurally different probes were incorporated at different
initial rates at the N termini of the two proteins tested, RHO and CAT.
The time course of peptide formation indicates that nearly linear
incorporation of [3SS]Met proceeded for about 3-5 min
and highest incorporation of [35S]Met at the N terminus
of RHO or CAT peptides occurred when protein synthesis was initiated
with f[35S]Met-tRNAf. With
fluorophore-Met-tRNAf, a brief lag period was observed. At
the 5-min time point, the percentage incorporation of the various
probes attached covalently to the [35S]methionine ranged
from 30% to 62% of the level reached with formyl-methionine. With
both plasmids containing either the RHO or the CAT coding sequence, the
order of reduced initiation was identical; pyrene-Met-tRNAf
had the least and eosin-Met-tRNAf had the largest effect.
In unpublished experiments,3 we noted that the
binding of eosin-Met-tRNAf to salt-washed ribosomes was
reduced compared with binding of fMet-tRNAf.
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Fig. 2.
Kinetics of initiation of RHO or CAT peptides
synthesized with different fluorophore-Met-tRNAf
species. RHO (panel A) or CAT
(panel B) was synthesized in vitro in
a coupled transcription/translation assay using 5 µl of S30/30 µl
of reaction mixture. Synthesis was initiated by
N-formyl-[35S]Met-tRNAf ( ),
cascade yellow-[35S]Met-tRNAf ( ),
pyrene-[35S]Met-tRNAf ( ),
coumarin-[35S]Met-tRNAf (×), or
eosin-[35S]Met-tRNAf (*). Aliquots, withdrawn
at the indicated times, were trichloroacetic acid-precipitated, and
their radioactivity was determined. The results are expressed for
30-µl samples.
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Earlier observations indicated that in the coupled
transcription/translation system used here, polypeptide synthesis
(mainly elongation after the initial 5 min) proceeds nearly linearly
for about 20 min, after which the rate declines rapidly and approaches zero after 30 min of incubation at 37 °C (16). In the experiments leading to the data presented in Table I, incubation time was extended
to 30 min to allow maximum elongation of initiated peptides. The data
given in Table I indicate a reduction in the amount of enzymatically
active RHO or CAT, when fluorophore-Met formed the N terminus. For RHO,
this reduction is compatible with the data presented in Fig.
3 and may be explained by the differences in the rate of initiation shown in this figure. However, with CAT, a
larger decrease in enzymatic activity relative to the reduced incorporation of the fluorophore was measured. Therefore, we wanted to
determine whether the elongation was affected after nascent peptides
had been initiated with fluorophore-Met. For this reason, we analyzed
the pattern of nascent polypeptides with the results given in the
following section.
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Fig. 3.
Pause-site peptide pattern of RHO or
CAT. RHO (lanes 1-5) or CAT
(lanes 6-10) was synthesized using
N-formyl-[35S]-Met-tRNAf
(lanes 1 and 6), cascade
yellow-[35S]Met-tRNAf (lanes
2 and 7),
pyrene-[35S]Met-tRNAf (lanes
3 and 8),
coumarin-[35S]Met-tRNAf (lanes
4 and 9), or
eosin-[35S]Met-tRNAf (lanes
5 and 10) as initiator tRNA under optimal
translation conditions. The reaction mixtures were incubated at
37 °C for 30 min, then analyzed by SDS-PAGE and
phosphorimaging.
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The Pattern of RHO and CAT Pause-site Peptides--
The
pattern of synthesized polypeptides of the two test proteins initiated
with fMet-tRNAf or each of the four forms of
fluorophore-Met-tRNAf was revealed after SDS-PAGE, followed
by phosphorimaging, as described under "Experimental Procedures."
The patterns of peptides radioactively labeled from
[35S]Met in the initiator tRNA are shown for RHO and CAT
in Fig. 3. In case of RHO (lanes 1-5),
full-length protein is formed, although to different extent. It is the
most prominent band with all initiator tRNA species. In addition, a
ladder of discrete peptide bands is seen. These "pause-site
peptides" are in the size range of 5-33 kDa, the mass of full-length
RHO. These different bands represent points at which translation is
temporarily slowed or stopped. The cause of this pause in translation
has not been established. One of our objectives was to determine if
modification of the N terminus of the test proteins by attaching
different fluorophores affects the pattern of these pause-site
peptides. This does not seem to be the case, at least on the
qualitative level.
The results given in Fig. 3 for RHO confirm the reduction in overall
synthesis of polypeptides presented in Table I for fluorophore-Met-RHO. Synthesis of eosin-RHO was the lowest. The same is true for CAT (Fig.
3, lane 10). In addition, with CAT, there
is a remarkable reduction in full-length product formed when synthesis
was initiated with all fluorophore-Met-tRNAf. Full-length
CAT is reduced on the expense of small polypeptides of 6.5 kDa and
below, with the most prominent CAT peptides having a mass of 3.5-4 kDa
(Fig. 3, lanes 7-10). Pyrene-Met at the N
terminus of CAT exerts the strongest effect.
Accumulation of these peptides was evident in the time course of CAT
synthesis shown in Fig. 4. When CAT
synthesis was initiated with fMet-tRNAf (panel
A), polypeptides in the size range of about 4 kDa were
visible after 5 and 10 min of incubation but were reduced after longer
incubation. With cascade yellow- (panel B) or
pyrene-Met-tRNAf (panel C), these low
molecular mass polypeptides of 35-40 amino acids in length became the
predominant bands after 10 and 30 min of incubation as seen by
phosphorimaging after gel analysis.
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Fig. 4.
Kinetics of CAT synthesis initiated with
either fMet-tRNAf or
fluorophore-Met-tRNAf. CAT synthesis was initiated
with N-formyl-[35S]Met-tRNAf
(panel A), cascade
yellow-[35S]Met-tRNAf (panel
B), pyrene-[35S]Met-tRNAf
(panel C), or
eosin-[35S]Met-tRNAf (panel
D). Reaction mixtures were incubated, and, at indicated
times, aliquots were withdrawn and analyzed by SDS-PAGE and
phosphorimaging.
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The results with RHO were completely different (Fig.
5). After the initial 3 min of
incubation, the most prominent polypeptide appeared to be full-length
rhodanese regardless whether fMet, cascade yellow-Met, pyrene-Met, or
eosin-Met formed the N terminus.
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Fig. 5.
Kinetics of RHO synthesis initiated with
either fMet-tRNAf or
fluorophore-Met-tRNAf. The experiment was carried out
as described in the legend to Fig. 4, except that RHO was synthesized.
Panel A, synthesis with
N-formyl-[35S]Met-tRNAf; panel
B, synthesis with cascade
yellow-[35S]Met-tRNAf; panel
C, synthesis with
pyrene-[35S]Met-tRNAf; panel
D, synthesis with
eosin-[35S]Met-tRNAf.
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In summary, comparing fMet with fluorophore-Met at the N terminus, the
results show no major differences in the pattern of transient RHO
nascent peptides during the time of elongation, and full-length RHO is
efficiently formed. In the case of CAT, however, the rate of elongation
appears to be reduced when fluorophore-Met forms the N terminus. The
results demonstrate that fluorophores of different size and
hydrophobicity cause quantitative effects on ribosomal pausing at
pre-existing pause sites when CAT mRNA is translated. These effects
result in accumulation of fluorophore-Met-CAT peptides below 6 kDa.
It should be emphasized that the peptide patterns shown in Figs. 3-5
result from the radioactive methionine (to which different fluorophores
are covalently attached) at the N terminus of the polypeptides. To
demonstrate that these polypeptides were not generated by proteolytic
degradation of larger products or were the result of premature release
from the ribosomes, we carried out experiments in which the reaction
mixture after coupled transcription/translation (in the presence of
non-radioactive fMet- or pyrene-Met-tRNAf and
non-radioactive amino acids) was incubated with
[32P]C-puro. The experimental details are given under
"Experimental Procedures." The radioactively labeled polypeptides
were analyzed by SDS-PAGE and phosphorimaging. These results are given
in Fig. 6. It was observed that most
sizes of pause-site peptides seen in Fig. 3-5 reacted with puromycin.
Similar [32P]C-puro-peptide patterns were observed when
the other fluorophore-Met-tRNAf species initiated peptide
synthesis (data not presented). The results indicate that the low
molecular mass CAT peptides are not aborted and released peptides but
are nascent peptides bound to the ribosomal P site.
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Fig. 6.
Reaction of nascent peptides with
C-puro. RHO (lanes 1 and 2) or
CAT (lanes 3 and 4) synthesis was
initiated with unlabeled fMet-tRNAf (lanes
1 and 3) or unlabeled
pyrene-Met-tRNAf (lanes 2 and
4). Synthesis was carried out with 5 µl of S30 in the
reaction mixture during an incubation at 37 °C for 30 min.
Immediately after this incubation, [32P]C-puro was added
to the reaction mixtures and the incubation was continued as described
under "Experimental Procedures." The reaction products were
analyzed by SDS-PAGE and visualized by phosphorimaging.
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Nascent CAT Peptides Are in a Relatively Hydrophilic
Environment--
Fluorescence techniques were applied to study the
environment of the N-terminal probe on nascent CAT peptides. With
a sensitive probe, changes in quantum yield, in the maximum of the
emission spectrum, and in anisotropy will indicate interaction with
other molecules or changes in the environment.
CAT peptides were synthesized with pyrene-Met at their N terminus under
conditions in which low molecular weight nascent peptides accumulate on
the ribosomes, and then the ribosomal fraction was isolated (see
"Experimental Procedures") and subjected to fluorescence measurements. Fig. 7 (spectrum
1) shows the fluorescence emission spectrum of the
resuspended ribosomes bearing nascent pyrene-CAT peptides. This
fraction was then treated with C-puro (see "Experimental Procedures") and analyzed again in the fluorometer. The spectrum after puromycin reaction is shown by line 2 in
Fig. 7. A large increase in fluorescence intensity was observed. This
increase in quantum yield signals a move of the probe to a much
more hydrophobic environment. Calculated fluorescence parameters for
the spectra shown in Fig. 7 as well as anisotropy calculations are
compiled in Table II. Included in this
table are the corresponding values for pyrene-Met-tRNAf
free in buffered solution.
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Fig. 7.
Fluorescence spectra of ribosome-bound
pyrene-labeled CAT peptides and their release by puromycin.
Pyrene-labeled CAT was synthesized in the cell-free bacterial system
and after 30 min of incubation, the ribosomal fraction was obtained by
Airfuge centrifugation. The fluorescence spectrum of the resuspended
ribosomes bearing 4 pmol of CAT peptides was taken (spectrum
1). Then, 6 µl of C-puro was added to the same sample,
which was incubated for 10 min at 37 °C, followed by fluorescence
measurements (spectrum 2). The ribosomes were
separated by a second Airfuge centrifugation and fluorescence spectra
were recorded for both fractions, the resuspended ribosomes
(spectrum 3) and the supernatant fraction
(spectrum 4). Based on radioactivity, about 50%
of the puromycin-tagged CAT peptides were released. The spectrum of 5 pmol of free pyrene-Met-tRNAf is shown as comparison
(spectrum 5).
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Table II
Fluorescence properties of pyrene-labeled CAT and its release from
ribosomes by puromycin
The fluorescence parameters given here were derived from the spectra
for samples 1-4 as shown in Fig. 7. Emax,
wavelength of emission maximum; A, fluorescence anisotropy;
Rel Q, relative fluorescence quantum yield.
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After the measurements indicated in Fig. 7 (spectrum
2) were completed, the sample was centrifuged to analyze
whether the pyrene-CAT-C-puro peptides were separated from the
ribosomes. Both fractions, the resuspended ribosomes and the resulting
supernatant, were subjected to fluorescence measurements with the
results shown in Fig. 7 (spectra 3 and
4, respectively). The results indicate that the pyrene-CAT
peptides were released into the soluble fraction. The quantitative
fluorescence data for spectra 3 and 4 are included in Table II.
We interpret these results to indicate a change in the environment of
the pyrene moiety at the N terminus of the nascent CAT peptides from a
less hydrophobic (relatively hydrophilic) to a very hydrophobic
surrounding. We emphasize the less hydrophobic/relatively hydrophilic
aspect, because the relative fluorescence quantum yield of the nascent
pyrene-Met-CAT peptides on the ribosomes is appreciably higher than the
quantum yield of free pyrene-Met-tRNAf (Table II). Further,
we interpret the results to indicate a relatively hydrophilic
environment of the probe when the pyrene-CAT peptides are bound to the
ribosomes probably inside the tunnel of the 50 S ribosomal subunit as
discussed below. When released, the peptides may collapse with the
pyrene buried inside the peptide in a very hydrophobic pocket.
| |
DISCUSSION |
The fluorescent probes used in the present study differ in their
molecular size, shape, charge, and hydrophobicity. Computer-aided calculations of the dimensions of the probes indicated that the fluorophores used varied from 15 to 20 Å in length and about 5 Å in
diameter with the exception of eosin, which has a diameter of about 11 Å with 4 bromine atoms bonded to the xanthen ring system. All
fluorophore-Met-tRNAf molecules tested cause a reduction in
both CAT and RHO polypeptides formed, which in the case of RHO can be
traced to less efficient initiation. Eosin-Met-tRNAf has
the most pronounced effect.
In addition, the results presented above indicate that translational
pausing at some of the early pause sites is prominent when probes are
incorporated at the N terminus of CAT. Pyrene caused a greater increase
in pausing than coumarin in producing CAT nascent peptides of about
30-35 amino acids in length. A stronger effect than with coumarin is
also observed with cascade yellow, which is zwitterionic in nature. The
pyrene derivative appears to be the most hydrophobic (based on the HPLC
elution profile of the fluorophore-Met-tRNA) of the fluorophores
tested, although it is in a similar size range to cascade yellow and
coumarin. The results indicate that neither hydrophobicity nor the
ionic character of the probe is the unique cause of pausing at specific sites.
Low molecular weight pause-site peptides of CAT were observed
previously when protein synthesis was initiated with
coumarin-Met-tRNAf and elongation was slowed by reducing
the amount of the E. coli extract used in the assay (5). It
was suggested that the primary amino acid sequence at the N terminus
might be the determining element in the accumulation of pause-site
peptides. CAT with a very hydrophilic N terminus was assumed to be
strongly affected by the hydrophobic derivative of N-terminal Met
during the required folding of the nascent peptide to allow for its
path through the tunnel of the large ribosomal subunit (5). We wish to
modify this hypothesis, because similar experiments as reported above on the elongation of RHO and CAT were carried out with another protein, E. coli release factor RF-1. Its N-terminal amino
acid sequence (17) gives a hydrophobicity plot (18) very similar to
CAT; however, there was no accumulation of low molecular weight nascent
peptides, when RF-1 synthesis was initiated with pyrene- or cascade
yellow-Met-tRNAf (data not presented).
What causes CAT peptides to accumulate when pyrene, cascade yellow, or
coumarin forms the N terminus? It should be pointed out that an
N-terminal probe on nascent CAT peptides became accessible to specific
antibodies only when a mass of 8.5 kDa was reached. The corresponding
values for RHO and for MS2 coat protein were 6 and 4.5 kDa,
respectively (4). The nascent CAT peptides inside the ribosomal tunnel
may form a more compact structure, which is affected by the bulky
N-terminal extension in contrast to the secondary structures acquired
by the other proteins tested. The crystal structure of monomeric CAT
(19) is very compact with no formation of domains as is the case with
RHO (20). The crystal structure of RF-1 is not known yet. Secondary
structure prediction according to Garnier and Robson (21) indicates an
-helical structure for the first 40 amino acids of RF-1. The N
terminus of RHO is quite flexible; then an -helix is formed by amino
acids 11-22. The crystal structure of CAT indicates an
N-terminal 
-sheet (amino acids 1-12), followed by a short -helix
before another -sheet is formed. We do not know whether these
differences in the N-terminal secondary structures are the cause for
the effects seen when pyrene- or cascade yellow-Met are at the N
terminus of the nascent peptides. We assume this might be the case.
An important conclusion from the results presented above is that the
N-terminal pyrene on the nascent CAT peptides is in a relatively
hydrophilic environment. The most prominent CAT peptides are in the
range of 4 kDa: about half the mass before the N terminus becomes
accessible to antibodies. The recent model of the 70 S ribosome
developed from image reconstruction of electron micrographs (22)
confirms a structured tunnel through the 50 S subunit. The images at a
resolution of 15 Å position the CCA end of P-site bound tRNA at the
mouth of the tunnel, which is assumed to be the conduit for the nascent
polypeptides. We estimated the length of the tunnel to be about 70 Å and its width between 16 and 19 Å, based on these cryoelectron
microscopy data together with recently published crystallographic data
on the 50 S subunit (23). Combining these numbers with the secondary
structure for the N-terminal 40 amino acids of the CAT still leaves the
question open where the N-terminal pyrene is localized within the
tunnel. Recent data (24) obtained with an N-terminal photo-activatable
probe on different nascent polypeptides indicated cross-linking to
specific nucleotides of the 23 S RNA. When the synthesized peptides had a length of about 30 amino acids, cross-linking to several nucleotides of the ribosomal RNA was observed. For all three polypeptides of this
length (ompA peptide, tetracycline resistant gene-peptide, and T4 gene
60 peptide), cross-linking to nucleotides 2062 and nucleotide 2609 was
found consistently; additional cross-links were determined with the
second peptide. These results are quite remarkable; they show that
different peptides of the same length gave different cross-links.
Furthermore, nucleotide 2609 is in domain V, the peptidyltransferase
center. This finding indicates that nascent peptides are not traversing
the ribosomal tunnel in a linear arrangement. Most importantly, the
results suggest a hydrophilic environment that the nascent peptide may
encounter inside the ribosomal tunnel. Our fluorescence results with
nascent pyrene-CAT peptides support this notion.
| |
ACKNOWLEDGEMENTS |
We appreciate the initial gift of
cytidylic acid-puromycin from Dr. Rachel Green (Department of Molecular
Biology and Genetics, Johns Hopkins University, Baltimore, MD). We
thank Suleyman Bahceci (Department of Chemistry and Biochemistry,
University of Texas, Austin, TX) for calculating the dimensions of the
probes by Molecular Mechanics software. In addition, we thank Delbert
Brod for excellent technical assistance and Barbara Jann for help with
the typescript.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM 53152 and Welch Foundation Grant F 1348.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.
To whom correspondence should be addressed. Tel.: 512-471-5874;
Fax: 512-471-9686; E-mail: b.hardesty@mail.utexas.edu.
2
S. Seliger, O. W. Odom, G. Kramer, and B. Hardesty, unpublished data.
3
B. McIntosh, V. Ramachandiran, G. Kramer, and B. Hardesty, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
fMet-tRNAf, E. coli initiator tRNA aminoacylated
with methionine, the amino group of which is formylated;
CAT, chloramphenicol acetyltransferase;
C-puro, cytidylic acid-puromycin;
DTSSP, 3,3-dithiobis(sulfosuccinimidyl)propionate;
HPLC, high
performance liquid chromatography;
PAGE, polyacrylamide gel
electrophoresis;
RHO, rhodanese;
Tricine, N-tris(hydroxymethyl)methylglycine.
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