(Received for publication, April 25, 1997)
From the Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
Escherichia coli trigger factor has prolyl-isomerase and chaperone activities and associates with nascent polypeptide chains. Trigger factor has a binding site on ribosomes, which is a prerequisite for its efficient association with nascent chains and its proposed function as a cotranslational folding catalyst. We set out to identify the domain of trigger factor that mediates ribosome binding. Of a series of recombinant fragments, the amino-terminal fragments, TF (1-144) and TF (1-247), cofractionated with ribosomes from cell extracts and rebound to isolated ribosomes in vitro. They competed efficiently with full-length trigger factor for stoichiometric binding to a single site on the large ribosomal subunit. However, TF (1-144) and TF (1-247) differed from full-length trigger factor in that their association with ribosomes was not strengthened by the presence of nascent chains, indicating a role for carboxyl-terminal trigger factor segment in sensing the translational status. The domain responsible for ribosome binding was further investigated by limited proteolysis of recombinant fragments. A stable domain comprising the amino-terminal 118 residues was identified that was still capable of ribosome binding and thus represents a novel structural and functional element of trigger factor.
Trigger factor was first identified in Escherichia coli (1) but meanwhile has also been found in other bacteria (2). It was isolated by its activity to promote in vitro the translocation of precursors of the outer membrane protein A (proOmpA) into membrane vesicles (3, 4). Trigger factor is associated with ribosomes prepared from cell extracts and binds to purified large ribosomal subunits (5) known to contain the exit site for nascent polypeptide chains. These findings led to the earlier proposal that trigger factor acts as a secretion-specific chaperone to shuttle precursors of secretory proteins from their place of synthesis to membrane translocation sites (5). However, E. coli cells depleted of trigger factor do not show secretion defects (6).
Recently, trigger factor was identified as a ribosome-associated peptidyl-prolyl-cis/trans-isomerase (PPIase)1 (7). The purified protein catalyzed the prolyl isomerization-dependent refolding of the unfolded protein substrate RNaseT1 much more efficiently than other PPIases tested before (7). This high efficiency is due to the cooperation of PPIase and chaperone activities within the protein (8). In independent studies, using in vitro translation systems, trigger factor was identified as a major cross-linking partner for nascent polypeptide chains of cytoplasmic and secretory proteins (9, 10). The association of trigger factor with ribosomes was found to be sensitive to the translational status. Translating ribosomes formed complexes with trigger factor that were resistant to high salt treatment and disrupted by puromycin-mediated release of the nascent polypeptide chains, whereas nontranslating ribosomes formed salt-sensitive complexes only (10). Together, a scenario was proposed in which trigger factor, by virtue of its PPIase activity and an additional chaperone-like function, assists the folding of nascent polypeptide chains as they emerge from the ribosome (7, 9, 10). In addition, trigger factor functionally cooperates with the GroEL chaperone, as shown by its activity to promote the GroEL-dependent degradation of polypeptides in vivo (11).
The PPIase activity was localized in a central domain of trigger factor between amino acids 145 and 247/251 (12, 13). This assignment was predicted on the basis of a sequence similarity to FK506 binding protein (FKBP)-type PPIases (10, 14) and a hydrophobic cluster analysis (14) and experimentally verified by limited proteolysis of the native protein (12, 13). The isolated PPIase domain was ~1000-fold less active than full-length trigger factor in refolding of RNaseT1, indicating that the flanking amino- and/or carboxyl-terminal parts of its polypeptide chain are required for high refolding activity (8).
We now set out to identify the domain of trigger factor that mediates ribosome binding. This domain is likely to be central to a putative mechanism that targets trigger factor to nascent polypeptide chains. Assuming a modular structure of the protein, we designed recombinant fragments of trigger factor on the basis of the structural information obtained by limited proteolysis. They comprise the amino-terminal part of the polypeptide chain, TF (1-144), the central FKBP domain, TF (145-247), the carboxyl-terminal portion, TF (248-432), or combinations thereof, TF (1-247) and TF (145-432). We found that the amino-terminal 118 amino acids of trigger factor as part of TF (1-144) are necessary and sufficient for specific ribosome binding.
Plasmid pTIG2 (6) containing the full-length, wild-type
tig gene from E. coli served as a template for
the polymerase chain reaction-based amplification of different gene
fragments. The gene fragment corresponding to the first 144 codons of
tig was amplified using the primer pair 5-GGC CGG ATC CAT
GCA AGT TTC AGT TGA AA-3
(P1) and 5
-GGC CGG ATC CCA GAG TAT CCA GCA
TGC CG-3
(P4), the fragment corresponding to codons 145-247 using the
primer pair 5
-GGC CGG ATC CCG TAA ACA GCA GGC GAC CT-3
(P2) and
5
-GGC CGG ATC CTT CCG GCA GTT CAC GCT CT-3
(P5), and the fragment
corresponding to codons 248-432 using the primer pair 5
-GGC CGG ATC
CCT GAC TGC AGA ATT CAT CA-3
(P3) and 5
-GGC CGG ATC CCG CCT GCT GGT
TCA TCA GC-3
(P6). Fragments corresponding to codons 1-247 and
145-432 were amplified with the primer pairs P1/P5 and P2/P6,
respectively. A gene fragment encoding the amino-terminal 118 amino
acids of trigger factor was polymerase chain reaction-amplified using
the primers P1 and 5
-GGC CGG ATC CCT CGA GTT CAA CTT CCG GA-3
(P7).
Polymerase chain reactions were carried out with Pwo DNA polymerase
(Boehringer Mannheim) according to the manufacturer's instructions.
Amplification products were purified, digested with BamHI,
and ligated into the single BamHI site of the expression vector pDS56 RBSII, 6 × His (15). The resulting expression
products have four additional amino acids at the amino terminus (MRGS) and eight additional amino acids at the carboxyl terminus (RSHHHHHH). Ligations were transformed into E. coli DH5
containing
pDMI,1 which encodes lacIq (16) followed by
sequencing of the inserts. Clones with wild-type tig
sequences were used for expression and purification of trigger factor
fragments. A construct for the expression of full-length, His-tagged
trigger factor was obtained by ligation of the two overlapping
polymerase chain reaction-derived gene fragments at the single
AccI restriction site.
For purification of the proteins, expression of the trigger factor
fragments in DH5 containing pDMI,1 was induced at an
A600 of 0.5 with 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
30 °C in LB medium. Cells were harvested by centrifugation in the
cold and resuspended in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mg/ml lysozyme, and 15%
(w/v) sucrose, pH 7.6 at 4 °C). Following incubation at 4 °C for
15 min, the cells were frozen at
80 °C, thawed slowly on ice, and
lyzed by the addition of a 4-fold volume of cold distilled water and
brief sonication. After centrifugation (30,000 × g for
30 min at 4 °C), the cleared supernatants were loaded onto
Ni2+-NTA agarose (Qiagen) columns equilibrated in buffer A
(20 mM Tris-HCl, 2 mM 2-mercaptoethanol, pH 7.6 at 4 °C) and 100 mM NaCl. Columns were washed with three
column volumes buffer A containing 100, 200, and 500 mM
NaCl, followed by step elution with 1 column volume each of 10, 20, 30, 40, 50, 100, and 200 mM imidazole in buffer A and 100 mM NaCl. Trigger factor peak fractions were either pooled
and dialyzed against trigger factor storage buffer (20 mM
Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, and 2 mM 2-mercaptoethanol) or 4-fold diluted with distilled
water and rechromatographed on a Protein Pak Q 8 HR (Waters) strong
anion exchange column, essentially as described before, for native
full-length trigger factor (12). Purity of the individual fragments was
assessed by SDS-PAGE analysis and Coomassie Brilliant Blue staining.
Protein concentrations were determined using the BCA-kit (Pierce) and
bovine serum albumin as a standard.
DH5 derivatives expressing the different trigger factor
fragments were cultured in LB medium at 30 °C to an
A600 of 0.5. Low level expression of trigger
factor fragments was achieved by the addition of
isopropyl-1-thio-
-D-galactopyranoside to 20 µM for 1 h. 20-ml aliquots of the cultures were then
rapidly cooled to 0 °C in an ice-water bath and centrifuged at
4 °C to harvest the cells. Cells were lyzed essentially as described
above except that the Mg2+ concentration was permanently
kept at 10 mM. Crude lysates were cleared by centrifugation
(15,000 × g for 15 min at 4 °C) and layered onto
sucrose cushions (20% (w/v) sucrose in 20 mM Tris-HCl, 100 or 500 mM KCl, 10 mM MgCl2, and 5 mM 2-mercaptoethanol, pH 7.6 at 4 °C; three volumes
cushion per volume of lysate). Ribosomes were pelleted at 213,000 × g for 1 h at 4 °C in a TLA-100 rotor (Beckman).
Ribosomal pellets were resuspended in SDS-PAGE sample buffer, and
aliquots of ribosomes and postribosomal supernatant (50% of the
corresponding amount of ribosomal pellets) were analyzed on 13.5%
acrylamide denaturing gels. Gels were blotted onto nitrocellulose, which was then probed with a trigger factor-specific antiserum from
rabbit and a chemiluminescence-based detection system (BM blotting
substrate, Boehringer Mannheim).
Ribosomes were
purified from a derivative of the E. coli C600 strain.
Briefly, an inducer-regulated chromosomal tig allele was
introduced from strain BG87 (6) into C600 by P1vir
transduction and selection of Ampr transductants.
Transductants show arabinose-dependent expression of the
chromosomal tig gene. To prepare ribosomes depleted of endogenous trigger factor, C600 transductants were grown in the absence
of arabinose. Ribosomes were purified as described before (10),
omitting, however, the high salt wash step, and stored in ribosome
buffer (10 mM Tris-HCl, 6 mM MgCl2,
60 mM NH4Cl, and 4 mM
2-mercaptoethanol, pH 7.6) at 80 °C. Analytical sucrose gradient
centrifugation showed a monosome:subunit ratio of 70:30%. Trigger
factor fragments were added at the indicated concentrations followed by
60-min incubation at 30 °C. Samples were then layered onto a 3-fold
volume of 20% (w/v) sucrose in buffer A (100 mM KCl, see
above). Following high speed centrifugation as described in the
previous section, supernatants and pellets were separated and analyzed
by SDS-PAGE and Coomassie Brilliant Blue staining. For sucrose gradient
centrifugation, four A260 units of preformed ribosome-TF (1-144) complexes were loaded onto 12 ml of 10-30% (w/v)
linear sucrose gradients in buffer B (20 mM Tris-HCl, 100 mM NH4Cl, and 5 mM
2-mercaptoethanol, pH 7.6) containing 1 mM MgCl2 (subunit dissociation conditions) or 6 mM
MgCl2 (subunit association conditions). Gradients were run
in a Beckman SW40 Ti rotor at 36,000 rpm for 100 min at 3 °C
followed by fractionation from the tube bottom (40 fractions of 300 µl each). SDS-PAGE analysis was done on 13.5% (w/v) acrylamide
denaturing gels, followed by blotting onto nitrocellulose filters.
Filters were probed with antisera raised against trigger factor or
ribosomal proteins S7 and L7/L12 and detected as before.
For analysis by SDS-PAGE, 40 µg of trigger factor fragments were incubated with 80 ng of proteinase K (Boehringer Mannheim) in a final volume of 200 µl of 10 mM Tris-HCl (pH 8), 1 mM CaCl2 at 30 °C. At the time points indicated, aliquots were taken, mixed with phenylmethylsulfonyl fluoride (final concentration, 1 mM) and analyzed by SDS-PAGE (13.5% (w/v) acrylamide) followed by silver staining. For amino-terminal sequencing, digested protein was separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes (Millipore) and microsequenced on a 473A Sequencer (Applied Biosystems). For mass spectroscopy, 100 µg of TF (1-144) were digested with proteinase K for 20 min at 30 °C, mixed with phenylmethylsulfonyl fluoride and passed over a protein C18 reversed phase high-performance liquid chromatography column (Vydac). Mass spectroscopy was carried out on a Kratos Compact MALDI 3 version 4.0. For ribosome binding studies, TF (1-247) at a final concentration of 30 µM was digested with 6 nM proteinase K for 20 min at 30 °C, followed by the addition of phenylmethylsulfonyl fluoride to a 1 mM final concentration.
MiscellaneousPPIase activity assays were carried out at 10 °C using the substrate Suc-Ala-Phe-Pro-Phe-pNA essentially as described (10). The concentration of trigger factor fragments in the assay was 50 nM. For gel filtration analyses, volumes of 200 µl of the recombinant fragments (final concentration, ~20 µM) were loaded on a Superdex 75 column (10/30, Pharmacia Biotech Inc.) and eluted at a flow rate of 0.5 ml/min in buffer A, 100 mM NaCl. The column was calibrated with bovine serum albumin (67 kDa), ovalbumin (45 kDa), and lysozyme (14 kDa) as standards and blue dextran 2000 to detect the void volume. In vitro transcription/translation reactions of the lacZ gene were carried out as before (10).
The
design of trigger factor fragments is based on previous results
obtained by limited proteolysis of the full-length protein. Proteolysis
by proteinase K, endoproteinase Glu-C (V8), and subtilisin generated
stable fragments of ~12 kDa (amino acids 145-247/251) displaying
full PPIase activity (12, 13). Based on this information, we prepared
recombinant trigger factor fragments comprising the amino-terminal
segment (TF 1-144), the central PPIase domain (TF 145-247), the
carboxyl-terminal segment (TF 248-432), the overlapping fragments TF
(1-247) and TF (145-432), and full-length trigger factor, TF (1-432)
(Fig. 1). Each of these proteins carries
at its carboxyl terminus a His-tag to facilitate purification. High level expression of the individual fragments in DH5 host cells did
not perturb cell growth and resulted in predominantly soluble recombinant fragments. Proteins were purified by affinity
chromatography on Ni2+-NTA agarose and, depending on
the purity achieved, a second chromatography step on a strong
anion exchange column (Fig. 1).
To analyze the structural integrity of the fragments, PPIase activity assays, gel filtration analyses, and limited proteolysis were carried out. All fragments possessing the central FKBP domain displayed PPIase activity toward the oligopeptide substrate Suc-Ala-Phe-Pro-Phe-pNA. The specific activities were in the same range as reported before by us and others (7, 12, 13) for wild-type trigger factor and the isolated FKBP domain (Table I). Gel filtration analyses on a Superdex 75 column (linear separation range for globular proteins between 3 and 70 kDa) was carried out to further exclude that the purified fragments were misfolded and aggregated. For all fragments, 70% or more of the protein eluted within the included volume of the gel filtration column corresponding to monomeric or potentially dimeric states (Table I). Fragments containing the carboxyl-terminal domain, TF (1-432), TF (145-432), and TF (248-432), showed a tendency to aggregate at protein concentrations above ~10 µM, as indicated by a partial elution of these fragments in the void volume of the gel filtration column. Consistent with a previous report (13), the isolated PPIase domain eluted in a single sharp peak corresponding to a molecular mass 27 kDa. The result of limited proteolysis by proteinase K of full-length His-tagged trigger factor was indistinguishable from that observed before for untagged trigger factor (12). The central FKBP domain was entirely resistant to proteolysis in all fragments (data not shown; Fig. 7). The carboxyl-terminal domain of trigger factor, both as the isolated fragment TF (248-432) or as part of TF (1-432) and TF (145-432), was rapidly degraded without populating stable degradation intermediates (data not shown), as observed before for the untagged full-length protein (12, 13). The results of limited proteolysis of TF (1-144) and TF (1-247) are fully compatible with native polypeptide conformations and are detailed below (Fig. 7). Taken together, the purified recombinant trigger factor fragments, by three different criteria, display native or native-like conformations, allowing functional analyses. Caution may be required when interpreting results for the isolated carboxyl-terminal fragment, TF (248-432).
|
TF (1-247) and TF (1-144) Copurify with Ribosomes Prepared from Cell Extracts
To identify the domain of trigger factor that
mediates ribosome binding, we first investigated a potential
cosedimentation of the recombinant trigger factor fragments with
ribosomal particles prepared from cell extracts. The positive binding
controls were ribosomes prepared from DH5 cells, which chromosomally
encode untagged full-length trigger factor. At 100 mM KCl,
30-40% of the cellular trigger factor pool cofractionated with
ribosomes (Fig. 2). An increase of the
ionic strength to 500 mM during the centrifugation through
the dense sucrose cushion reduced the amount of cofractionating trigger
factor 2-3-fold. Of the two larger fragments, TF (1-247) but not TF
(145-432) cosedimented with ribosomes. Consistent with the failure of
TF (145-432) to bind to ribosomal particles, neither of its
subfragments TF (145-247) and TF (248-432) showed a ribosomal
localization. By contrast, TF (1-144), the amino-terminal domain of
trigger factor, cofractionated with ribosomes at 100 mM
KCl, indicating that this fragment is necessary and sufficient for
binding of trigger factor in vivo. However, binding of TF
(1-247) and TF (1-144) to ribosomal particles was more susceptible to
salt-induced dissociation than binding of the full-length protein.
TF (1-247) and TF (1-144) Rebind to Purified Ribosomes in Vitro
The ribosome-binding properties of the recombinant trigger
factor fragments were further analyzed in vitro with
purified ribosomes. To remove the endogenous trigger factor, ribosomes
were prepared from trigger factor-depleted cells. This approach avoided
high-salt washing of the ribosomes, which may cause microheterogeneity
of the ribosomal particles. The ribosomal preparation used was mainly composed of 70S monosomes, as revealed by analytical sucrose gradient centrifugation at 6 mM Mg2+ (data not shown).
Ribosomes were then incubated to equilibrium with a molar excess of
trigger factor fragments and reisolated by centrifugation through
sucrose cushions. Analysis of the ribosomal pellets and postribosomal
supernatants by SDS-PAGE confirmed the previous observation that TF
(1-247) and TF (1-144), but not TF (145-432), can bind to ribosomes
(Fig. 3). Neither TF (145-247) nor TF
(248-432) showed ribosome binding in vitro (data not
shown). TF (1-144) is thus the smallest of the trigger factor
fragments tested to bind to ribosomes in vivo and in
vitro. Judged from the staining intensities (Fig. 3), binding of
TF (1-247) and TF (1-144) to ribosomal particles shows an apparent
saturation at a 1:1 stoichiometry indicative of a single ribosomal
attachment site.
Trigger Factor and Its Subfragments TF (1-247) and TF (1-144) Compete for Binding to a Single Ribosomal Site
To substantiate
the observation that ribosome binding by trigger factor is a function
of its amino-terminal domain, direct competition experiments were
carried out. Ribosomes were incubated to equilibrium with equimolar
amounts of TF (1-432), TF (1-247), and TF (1-144), re-isolated by
centrifugation, and analyzed by SDS-PAGE (Fig.
4). In agreement with the experiment
shown in Fig. 3, visual inspection of the gel reveals that binding of
the individual trigger factor fragments to ribosomes occurs at an
apparent 1:1 stoichiometry. Co-incubation of equimolar, saturating
amounts of TF (1-432) and TF (1-144) or TF (1-432) and TF (1-247)
with ribosomes resulted in a ~50% reduction in binding of the
individual fragments and a concurrent appearance of the proteins in the
supernatant fractions. Moreover, a 2-fold molar excess of trigger
factor or trigger factor fragments during the incubation with ribosomes resulted in an equivalent binding to the ribosomal particles and appearance of unbound protein in supernatant fractions (Fig. 4). These
results indicate that TF (1-432), TF (1-144), and TF (1-247) have
the same binding specificity and apparent affinity toward a single
ribosomal attachment site.
TF (1-144) Binds to the Large Ribosomal Subunit
The
ribosomal binding site of trigger factor was shown earlier to map to
the large ribosomal subunit (5). Direct competition of TF (1-144) and
TF (1-432) for ribosome binding suggests that the amino-terminal
fragment is capable of discriminating the ribosomal subunits. This was
investigated by sucrose gradient centrifugation. We envisioned that
free TF (1-144) may smear into the 30S area of the sucrose gradients,
thereby mimicking binding to the small ribosomal subunit. To avoid this
potential difficulty, we used as the starting material for the gradient
centrifugations the ribosome-TF (1-144)-complex isolated by sucrose
cushion centrifugation in the presence of high magnesium concentration.
Four A260 units of this complex were centrifuged
in sucrose gradients containing either high or low magnesium
concentrations to stabilize or destabilize, respectively, the coupled
state of the ribosome. Fig. 5 shows the
distributions of TF (1-144), ribosomal S7 (to identify the small
ribosomal subunit), and ribosomal L7/L12 (to identify the large
ribosomal subunit) in the gradient fractions. Accordingly, TF (1-144)
binds to 70S couples (upper panel, fractions 15 and 16) and
50S large ribosomal subunits (upper panel, fractions 11 and
12; lower panel, fractions 10-12). There was no binding to the small ribosomal subunit under both magnesium conditions (both panels, fractions 7 and 8). Thus, TF (1-144) further resembles the full-length protein in that it is capable of discriminating the
ribosomal subunits and binding to large subunits only.
TF (1-144) and TF (1-247) Are Incapable of Forming Salt-resistant and Puromycin-sensitive Complexes with Translating Ribosomes in Vitro
In an earlier study, we found that the association of
trigger factor with ribosomes becomes salt-resistant when translation occurs (10). These complexes were disrupted by puromycin-mediated release of the nascent polypeptide chains (10). Both observations are
consistent with a scenario in which trigger factor, by hydrophobic interaction, binds to nascent polypeptide chains to assist their folding. Here, we tested whether TF (1-144) and TF (1-247) are capable of forming salt-resistant complexes with translating ribosomes. Trigger factor fragments were added at a final concentration of 1 µM to lacZ in vitro transcription/translation
reactions. The endogenous full length trigger factor of the cell-free
system was calculated to be present at a concentration of 0.2-0.4
µM during transcription/translation (5) and served as a
positive binding control. Fig. 6 shows
the presence of trigger factor and its fragments in the respective
ribosomal fractions. Accordingly, TF (1-144) and TF (1-247) differ
from full-length trigger factor in that their binding to ribosomes at
low salt concentration is insensitive to puromycin treatment. In
addition, these fragments cannot form salt-resistant complexes with
translating ribosomes as observed for the full-length protein. We
conclude that the amino-terminal fragment with or without the adjacent
FKBP domain is insufficient to allow salt-resistant and
puromycin-sensitive binding to ribosomes in the
transcription/translation assay. Neither of the fragments TF
(145-432), TF (145-247), and TF (248-432), even at concentrations
higher than 1 µM, showed significant cofractionation with
ribosomes in the transcription/translation assay (data not shown).
The Amino-terminal 118 Amino Acids of Trigger Factor Form a Compactly Folded Domain
We previously used limited proteolysis by proteinase K and endoproteinase Glu-C (V8) to dissect structural domains of trigger factor (12). Using the full-length protein, we failed to populate stable fragments except for the central FKBP-type PPIase domain. In the course of limited proteolysis of recombinant trigger factor fragments by proteinase K, however, we noticed the appearance of distinct products for TF (1-247) and TF (1-144) (Fig. 7). Within the first 2 min of digestion, TF (1-247) was degraded into two prominent proteolytic fragments (PK-1 and PK-3). By amino-terminal sequencing, PK-1 was identified as an extended PPIase domain starting with glycine 119. We had noticed this cleavage site before when sequencing a transient proteinase K fragment of native, full-length trigger factor (12). With time, this species was further trimmed amino-terminally to give the ~12-kDa FKBP fragment (PK-4) starting at arginine 145. PK-3 started with the amino acids MRGSMQVSV and, therefore, represents the amino terminus of TF (1-247). A comigrating species (PK-6), starting with the same amino-terminal amino acids, was generated by proteolysis of TF (1-144). Thus, amino acids 1-144 of trigger factor contain a compactly folded domain. To determine the carboxyl terminus of this domain, mass spectroscopy was performed with TF (1-247) and TF (1-144) samples that had been digested with proteinase K and passed over a reversed-phase HPLC column. The predominant proteolytic fragment common to TF (1-144) and TF (1-247) had an experimentally determined molecular mass of 13,497-13,498 Da. Taking into account that the vector encoded four additional amino acids at the amino terminus, this mass perfectly fits to a fragment of trigger factor ending with glutamine 118 and having a theoretical mass of 13,498.6. This is in agreement with the sequencing result for the larger proteolytic fragment of TF (1-247), PK-1, starting with glycine 119. A second proteolytic fragment (PK-2 and PK-5) was common to TF (1-247) and TF (1-144). Surprisingly, these polypeptides, although migrating slower in SDS-PAGE than PK-3 and PK-6, arose from further proteolysis of PK-3 and PK-6 by six amino-terminal amino acids as revealed by amino-terminal sequencing and mass spectroscopy. Together, amino acids 1-118 (or 3-118) comprise a compactly folded domain of E. coli trigger factor.
TF (1-118) Is Capable of Binding to RibosomesTo test
whether the above described structural element of trigger factor,
encompassing the amino-terminal 118 amino acids, also represents a
functional entity, two experimental approaches were taken:
(a) TF (1-118) was constructed genetically and purified as
a recombinant fragment by Ni2+-NTA affinity chromatography;
and (b) a preparative digest of TF (1-247) was carried out
to produce the proteinase K fragments PK-1 (starting with glycine 119)
and PK-3 (ending with glutamine 118). TF (1-118) and the proteolytic
fragments of TF (1-247) were incubated with equimolar amounts of
purified ribosomes followed by sucrose cushion centrifugation to
re-isolate the ribosomal particles (Fig.
8a). SDS-PAGE analysis of the
ribosomal fractions by staining with Coomassie Brilliant Blue was
insufficient to identify the trigger factor fragments, because
they comigrated with ribosomal proteins. Therefore, these fractions
were additionally subjected to immunoblotting using a trigger
factor-specific antiserum (Fig. 8a). Positive
ribosome-binding signals were obtained for recombinant TF (1-118),
residual TF (1-247), and the PK-3 fragment, representing the
amino-terminal 118 amino acids of TF (1-247). In control incubations
lacking ribosomes, all fragments were recovered from the supernatant
fractions. Furthermore, PK-1 exclusively remained in the supernatant
fractions, even in presence of ribosomes. Thus, the presence of TF
(1-118) and PK-3 in pellet fractions demonstrates ribosome binding by
these fragments in vitro. Recombinant TF (1-118), in
addition, was capable of binding to ribosomes in vivo, as
demonstrated by its cosedimentation with ribosomal particles prepared
from cell extracts of TF (1-118) overproducing cells (Fig.
8b). We conclude that TF (1-118) not only structurally but also functionally fulfills the criteria of a novel trigger factor domain that mediates binding to ribosomes in vivo and
in vitro.
The present study led to the identification of the ribosome
binding site of trigger factor. We found that the amino-terminal trigger factor fragment TF (1-144): (i) is necessary and
sufficient for ribosome binding in vivo and in
vitro; (ii) shares the same binding site with
full-length trigger factor on the large ribosomal subunit; and
(iii) contains a compactly folded, protease-protected domain
comprising the amino-terminal 118 amino acids of trigger factor. This
proteolytic fragment was capable of binding to ribosomes in
vivo and in vitro and, therefore, represents the second
structural and functional element of trigger factor described so far
(Fig. 9). Unlike the central PPIase
domain, which belongs to the FKBP family of PPIases (10, 12, 14),
homology searches against updated data bases did not reveal any protein
with similarity to TF (1-118). These findings do not formally exclude
the existence of additional sites within trigger factor that contribute
to ribosome binding but by themselves are insufficient for binding to
ribosomes. Such sites would be difficult to detect experimentally.
Based on limited proteolysis, the amino-terminal ribosome-binding domain and the central FKBP domain are separated by an exposed 26-amino acid peptide linker that is accessible to proteinase K and endoproteinase Glu-C (V8) (this study and Ref. 12). The PhD program for prediction of secondary structure and surface accessibility in proteins (17) predicted a surface-exposed loop conformation for amino acids 118-136 of E. coli and Haemophilus influenzae trigger factor. With proteinase K, the initial proteolytic cleavage within trigger factor occurs between glutamine 118 and glycine 119, followed by rather slow cleavage between leucine 144 and arginine 145. We, therefore, envision this 26-amino acid linker to be part of an extended PPIase domain rather than being part of the ribosome binding domain. An extended PPIase fragment, starting with glycine 119, was observed before upon digestion of the full-length trigger factor (12). The functional significance of this extension is unclear. In our previous study, TF (1-118) was not observed as a stable proteinase K fragment for the following reason. TF (1-118) essentially comigrates in SDS-PAGE with the PPIase domain fragment TF (145-247). The overall resistance to proteolysis of TF (1-118) is lower compared with that of the FKBP domain, explaining why TF (1-118) escaped the detection in the former study, in particular because longer digestion times were used. Stoller et al. (13) noticed an amino-terminal fragment of ~14 kDa upon digestion of trigger factor by subtilisin. Interestingly, in their study, ribosome-bound trigger factor was completely resistant to subtilisin treatment, indicating the occurrence of substantial structural rearrangements upon ribosome binding or a protection from proteolytic attack by the ribosomal particle. In agreement with our work, the amino-terminal portion of trigger factor was also very sensitive to digestion by subtilisin (13).
Concerning the function of trigger factor at the ribosome, a crucial finding of this study is that a recombinant fragment comprising both the ribosome binding domain and the PPIase domain, TF (1-247), does not form puromycin-sensitive and salt-resistant complexes with translating ribosomes. For this function, apparently, the entire trigger factor molecule is required. Given that the salt-resistant complex of full-length trigger factor with translating ribosomes is disrupted by puromycin treatment, it is tempting to assume that a trigger factor-nascent chain interaction is the basis of the tight ribosomal association. Accordingly, salt-resistant binding of trigger factor to ribosomes would reflect a nonionic interaction with nascent polypeptide chains. Folding assistance could be achieved by a cooperative process involving the catalytic power of the PPIase domain and an additional high affinity substrate binding reminiscent of molecular chaperone action (8). The isolated PPIase domain is deficient in high affinity binding of protein substrates (8), which may account for the failure of TF (1-247) to interact with translating ribosomes in a salt-resistant and puromycin-sensitive fashion. High affinity substrate binding by trigger factor could rely on a functional interaction of the FKBP domain with the carboxyl-terminal portion of its polypeptide chain and, possibly, additional contributions by the amino-terminal domain. It is evident, however, that the amino-terminal trigger factor domain fulfills the important targeting function to a site on the large ribosomal particle that is in proximity to the emerging nascent chain. This targeting function is likely to be a prerequisite for the efficient association of trigger factor with nascent chains.
We thank H. Bujard for generous support throughout this study, A. Bosserhoff and R. Frank for peptide sequencing and mass spectroscopy, H. Göhlmann and R. Herrmann for DNA sequencing, and A. Buchberger for critical reading of the manuscript. Antibodies to ribosomal proteins S7 and L7/L12 were kindly provided by R. Brimacombe.