 |
INTRODUCTION |
Three translational initiation factors (IF1, IF2, and
IF3)1 are required for the
initiation of protein synthesis in Escherichia coli (1, 2).
During initiation, IF3 binds to the 30 S subunit and shifts the
equilibrium between the ribosome and its subunits toward dissociation
(3, 4). IF1 and IF2 bind to the 30 S·IF3 complex. The initiation
factor·30 S complex binds the mRNA and fMet-tRNA, resulting in
the formation of an unstable preinitiation complex. This complex is
converted into a stable initiation complex when the initiator tRNA has
been selected and codon-antidocon interaction occurs (5, 6). IF3 has
three major functions: 1) it binds to the 30 S subunit, preventing the
joining of 50 S subunits (1-3, 5); 2) it increases the affinity of IF1
and IF2 for the 30 S subunit and stimulates fMet-tRNA binding to the 30 S subunit by promoting the conversion of the preinitiation complex to
the initiation complex (7, 8); and 3) it proofreads the selection of
fMet-tRNA at an AUG initiation codon (9-14).
The chloroplast translational initiation factors are postulated to be
functionally analogous to their E. coli counterparts. Only
IF2chl and IF3chl from Euglena
gracilis have been purified (15, 16). Both of these factors are
nuclear-encoded proteins in this organism (17, 18). IF3chl
has been resolved into three forms,
,
, and
. The
form has
a molecular mass of about 34 kDa, whereas the
and
forms have
molecular masses of about 45 kDa (16). In contrast, E. coli
IF3 has a molecular mass of 20 kDa. IF3chl is active on
E. coli ribosomes.
A complete cDNA encoding E. gracilis IF3chl
has been cloned and sequenced (17). The molecular mass deduced from the
nucleotide sequence is 58 kDa, including a signal peptide of 130-140
residues required for localization to the chloroplast. The mature form of this factor (IF3chlM) can be divided into three parts
(Fig. 1). An NH2-terminal extension termed the head (Hd)
region encompasses the first 140 amino acids. This region contains a
proline-rich sequence followed by a (GX)12 motif and a
short acidic sequence. A middle region of about 180 amino acids shows
homology to prokaryotic IF3 and is referred to as the homology (H)
domain (19). Structural analysis of E. coli and
Bacillus stearothermophilus IF3 indicates that this region
will fold into two highly compact domains separated by a lysine-rich
linker (20-24). The COOH-terminal extension is referred to as the tail
(T) region. This 64-amino acid region is rich in glutamic acid residues
(17).
Previous studies have shown that both IF3chlM and the
homology domain, IF3chlH, are active in promoting the
dissociation of ribosomal subunits and in promoting initiation complex
formation on E. coli ribosomes using poly(A,U,G) as an
mRNA (19). However, IF3chlM is only 10-20% as active
as IF3chlH in promoting initiation complex formation on
chloroplast 30 S ribosomal subunits using mRNAs carrying natural
translational start sites for chloroplast mRNAs (19). These
observations suggest that sequences in the head and tail regions of
IF3chl down-regulate the activity of this factor in
initiation. In the present work, the roles of sequences in the head and
tail regions in affecting the activity of IF3chl have been
examined in more detail.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[35S]fMet-tRNA and
[14C]AcPhe-tRNA were prepared as described (25, 26). A
plasmid carrying the 5' untranslated leader region and the
translational start site of the E. gracilis chloroplast rbcL gene fused in-frame to an internal coding region of the
neomycin phosphotransferase gene was transcribed in vitro
providing the mRNA, mRbcN (27). E. coli
ribosomes, initiation factors, E. gracilis chloroplast 30 S
subunits, IF2chl, IF3chl, and
IF3chlH antiserum were prepared as described (16, 19,
28-31).
Induction and Purification of Various Derivatives of
IF3chl--
Qiagen pQE vectors were used to express
IF3chl or its derivatives carrying a His tag at the COOH
terminus. The regions of IF3chl to be expressed were
amplified by polymerase chain reaction using the cDNA clone
described previously (17) or a derivative of this plasmid as template.
Cells were grown and IF3chl derivatives were induced as
described previously (19). Induction times were as follows: 45 min for
IF3chlHdH, 20 min for IF3chlHT, and 2-3 h for
the remaining constructs. IF3chlHdH was purified as
described for IF3chlM (19). The other forms of
IF3chl were purified using the two-step purification
procedure developed for IF3chlH (19).
Binding of IF3chl to 30 S Subunits--
The
indicated concentrations of IF3chl and chloroplast 30 S
subunits were incubated in a total volume of 250 µl in 50 mM Tris-HCl, pH 7.8, 10 mM dithiothreitol
(DTT), 50 mM NH4Cl, and 10 mM
MgCl2 at room temperature for 5 min. The mixture was
applied to a 5-ml 10-30% linear sucrose gradient prepared in the same
buffer except that the concentration of Tris-HCl was reduced to 10 mM. Samples were subjected to centrifugation at 48,000 rpm
for 2 h in a Beckman SW50.1 rotor. Gradients were fractionated at
a flow rate of 1 ml/min. Fractions (100 µl) were collected from the
region of the gradient containing the 30 S subunits. Aliquots (50 µl)
of appropriate fractions were analyzed for the amount of
IF3chl present using an ELISA (32). A standard curve for
each derivative tested was determined in each experiment to allow the
amount of IF3chl present to be quantified.
Assay for Initiation Complex Formation--
The abilities of
IF3chl and its derivatives to promote initiation complex
formation with E. coli 70 S ribosomes using poly(A,U,G) were
assayed as described (19). The abilities of IF3chl and its
derivatives to promote initiation complex formation with chloroplast 30 S subunits and mRbcN were determined as indicated (19).
Proofreading Assay--
This assay has been modified from the
method described in Ref. 33 for E. coli IF3. A complex
carrying AcPhe-tRNA bound to chloroplast 30 S subunits
(AcPhe-tRNA·poly(U)·30 S) was formed by incubation of chloroplast
30 S subunits (10 pmol) with poly(U) (2.5 µg) and AcPhe-tRNA (4 pmol)
in a reaction mixture (50 µl) containing 50 mM Tris-HCl,
pH 7.8, 10 mM dithiothreitol, 50 mM NH4Cl, and 15 mM MgCl2. After
incubation at 37 °C for 30 min, the mixture was diluted 2-fold with
50 mM Tris-HCl, pH 7.8, and 50 mM
NH4Cl in the presence of different concentrations of
IF3chl or its derivatives. Mixtures were incubated for an
additional 5 min at 37 °C. The destabilization of the complex by
IF3chl was monitored following dilution with 1 ml of
prewarmed dilution buffer (50 mM Tris-HCl, pH 7.8, 10 mM dithiothreitol, 50 mM NH4Cl, and 7.5 mM MgCl2). These reaction mixtures were
incubated at 37 °C for 5 min. The amount of initiation complex
remaining was determined by a nitrocellulose filter binding assay (19).
A similar assay was also carried out using a complex formed with 30 S
subunits (10 pmol), poly(A,U,G) (2.5 µg) and fMet-tRNA (4 pmol).
 |
RESULTS |
Inhibitory Effects of the Head and the Tail Regions on the Activity
of the Homology Domain of IF3chl--
Previous studies
have shown that the mature form of IF3chl
(IF3chlM) is almost as active as the homology domain
(IF3chlH) in an assay that measures the ability of
IF3chl to promote the binding of fMet-tRNA to E. coli 70 S ribosomes using poly(A,U,G). However,
IF3chlM shows very poor activity in promoting the binding of fMet-tRNA to chloroplast 30 S subunits in the presence of an mRNA carrying the translational initiation region of a natural mRNA (19). This observation indicates that the head, the tail, or
both have a negative effect on the activity of the homology domain of
IF3chl. To investigate which region or regions of
IF3chlM are responsible for this inhibitory effect, the
activities of a series of derivatives of IF3chl containing
different parts of IF3chl (Fig.
1) were tested. These derivatives were
designed based on the structures of the two best characterized
prokaryotic IF3s (from E. coli and B. stearothermophilus). The IF3s from these organisms have very
similar overall three-dimensional structures (20-22). However,
B. stearothermophilus IF3 is 9 residues shorter than
E. coli IF3 at the NH2 terminus.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Derivatives of IF3chl.
A, the overall structures of E. coli IF3,
B. stearothermophilus IF3, and IF3chlM are
shown. The open area represents the homology domain. For
E. coli IF3, the striped area represents the 9 residues at the NH2 terminus. For IF3chl, the
black area represents the head, and the cross-hatched area represents the tail. B, the regions of
IF3chl present in each of the derivatives. The residues
encompassed in each construct are IF3M, 130-538; IF3HdH, 130-489;
IF3HT, 278-538; IF3erH, 278-476; IF3rH, 284-476; IF3srH, 293-476;
IF3sHT, 293-538; and IF3sHT/3, 293-498). The numbering is based on
the initiator Met as residue 1. The transit peptide is predicted to be
130-140 amino acids in length.
|
|
Chloroplast homologues of both these prokaryotic IF3s were prepared
(Fig. 1). IF3chlsrH is the homologue of B. stearothermophilus IF3, whereas IF3chlrH is the
homologue of E. coli IF3. These two forms of
IF3chl differ by 9 residues at the NH2
terminus. To test the effects of sequences in the
NH2-terminal extension, a derivative, IF3chlHdH, encompassing the homology domain and the entire
head region was prepared. To test the effects of sequences in the
COOH-terminal extension, a derivative, IF3chlsHT, covering
the homology domain and the entire tail region was prepared. Note that
this derivative of IF3chl begins at the position
corresponding to the start of the B. stearothermophilus
factor.
The induction of IF3chlsHT, like IF3chlM,
results in a significant decrease in cell growth, indicating that the
tail of IF3chl is quite toxic to the cell. The induction of
IF3chlHdH has less effect on cell growth, whereas the
expression of IF3chlsrH does not affect cell growth to an
appreciable extent (data not shown). Each derivative of
IF3chl was purified; the derivatives were estimated to be
90-95% pure in all cases (Fig. 2).

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 2.
SDS-PAGE analysis of the purity of each
IF3chl derivative. Samples were analyzed on a 12% gel
and the proteins visualized by Coomassie Blue staining. Lane
1, broad range prestained molecular weight markers; lane
2, 2 µg of IF3chlsrH; lane 3, 2.5 µg of
IF3chlrH; lane 4, 3 µg of
IF3chlHdH; lane 5, 4 µg of
IF3chlsHT; lane 6, 2.4 µg of
IF3chlerH; lane 7, 2.5 µg of
IF3chlsHT/3; lane 8, 2.5 µg of
IF3chlHT.
|
|
The activity of each construct in promoting the binding of fMet-tRNA to
E. coli 70 S ribosomes was examined. As indicated in Fig.
3A, all of these forms of
IF3chl were quite active in this assay.
IF3chlHdH and IF3chlsHT had slightly less
activity than IF3chlsrH but slightly more activity than
IF3chlM. These results indicate that the head and tail had
little effect on the activity of IF3chl when E. coli 70 S ribosomes and a synthetic mRNA, poly(A,U,G), were
used. These derivatives of IF3chl were then tested for the
ability to promote the binding of fMet-tRNA to chloroplast 30 S
subunits using an mRNA carrying the translational initiation region
of the rbcL gene. As shown previously and as indicated in
Fig. 3B, IF3chlM had only 15-20% of the
activity of the homology domain of IF3chl in this assay.
The effect of sequences in the head was assessed by comparing the
activity of IF3chlHdH with IF3chlsrH (Fig.
3B). The head region reduced the activity of the homology
domain by 2-fold, indicating that the head accounts for about half of
the reduction in activity seen with IF3chlM. To assess the
effect of sequences in the tail, the activity of IF3chlsHT
was tested. IF3chlsHT had about 30% of the activity seen
with IF3chlsrH (Fig. 3B), indicating that
sequences in the tail account for a little over half of the inhibitory
effect seen in IF3chlM.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibitory effects of the head and tail
regions on the activity of the homology domain of IF3chl.
A, activities of IF3chlsrH ( ),
IF3chlHdH ( ), IF3chlsHT ( ), and
IF3chlM ( ) in promoting initiation complex formation on
E. coli ribosomes. The activities of these factors were
measured by determining their abilities to stimulate the binding of
fMet-tRNA to E. coli ribosomes in the presence of
poly(A,U,G) as described in Ref. 19. A blank (0.05 pmol) representing
the amount of fMet-tRNA bound in the absence of IF3chl has
been subtracted from each value. B, stimulation of
initiation complex formation on chloroplast 30 S ribosomal subunits in
the presence of 10 pmol of mRbcN (27). A blank (0.1 pmol)
representing the amount of fMet-tRNA bound in the absence of
IF3chl has been subtracted from each value.
|
|
A Small Region of the Head Is Sufficient to Confer Its Full
Inhibitory Effect--
The results presented above indicate that
sequences in both the head and the tail of IF3chlM have a
negative effect on the ability of the homology domain to promote
initiation complex formation. Additional constructs were then prepared
to narrow down the inhibitory region in the head. IF3chlerH
(Fig. 1) covers the homology region and 15 residues of the head from
the NH2 terminus of IF3chlsrH (the B. stearothermophilus homologue) to the edge of
(GX)12-acidic motif (19). IF3chlrH, the
E. coli homologue, is 9 residues longer than
IF3chlsrH at the NH2 terminus (Fig. 1). The
induction of either IF3chlrH or IF3chlerH
retards cell growth indicating that their expression is toxic to
E. coli (data not shown). Both IF3chlrH and
IF3chlerH were purified (Fig. 2, lanes 3 and
6).
IF3chlrH and IF3chlerH were as active as
IF3chlsrH when tested on E. coli 70 S ribosomes
(Fig. 4A). However,
IF3chlrH and IF3chlerH, like
IF3chlHdH, had half the activity of IF3chlsrH when tested on chloroplast 30 S subunits (Fig. 4B). These
results indicate that only 9 residues in the NH2-terminal
extension measured from the B. stearothermophilus factor are
required to give the inhibitory effect of the entire head region. This
observation is quite surprising because IF3chlrH is the
same length at the NH2 terminus as E. coli IF3.
The activity of E. coli IF3 decreases markedly without the
NH2-terminal hexapeptide (34).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Small regions of the head are sufficient to
confer the full inhibitory effect of the head. A, activities
of IF3chlerH ( ) and IF3chlrH ( ) compared
with IF3chlHdH ( ) and IF3chlsrH ( ) in
promoting initiation complex formation on E. coli ribosomes. A blank (0.05 pmol) representing the amount of fMet-tRNA bound in the
absence of IF3chl has been subtracted from each value. B, stimulation of initiation complex formation on
chloroplast 30 S ribosomal subunits in the presence of 10 pmol of
mRbcN (27). A blank (0.1 pmol) representing the amount of
fMet-tRNA bound in the absence of IF3chl has been
subtracted from each value.
|
|
Effect of the Tail on IF3chlsrH and Additive Effects of
Sequences in the Head and Tail Regions--
As indicated in Fig.
3B, the tail region contributed about half of the negative
regulatory effect seen with the mature form of IF3chl.
Secondary structure analysis indicated that the tail probably contains
two long helices that have a high probability of forming a coiled-coil.
To gain further insight into which sequences in the tail might be
responsible for this result, a derivative was prepared
(IF3chlsHT/3) that contained about
of the
sequences in the tail encompassing residues through the first helix
(Fig. 1). The induction of IF3chlsHT/3 had little effect on
the growth of E. coli. IF3chlsHT/3 was purified
to greater than 95% purity (Fig. 2, lane 7).
IF3chlsHT/3 had essentially the same activity as
IF3chlsrH when E. coli 70 ribosomes are used. When chloroplast 30 S subunits and mRbcN were used,
IF3chlsHT/3 was as active as IF3chlsrH (Fig.
5B). This result indicates
that the last
of the tail region are essential for the
inhibitory effect of the tail.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of the tail on IF3chlsrH
and additive effects of sequences in the head and tail. A,
activity of IF3chlsHT/3 ( ) and IF3chlHT
( ) compared with IF3chlsHT ( ), IF3chlsrH
( ), and IF3chlM ( ) in promoting initiation complex
formation on E. coli ribosomes. A blank (0.05 pmol)
representing the amount of fMet-tRNA bound in the absence of
IF3chl has been subtracted from each value. B,
stimulation of initiation complex formation on chloroplast 30 S
ribosomal subunits in the presence of 10 pmol of mRbcN (27).
A blank (0.1 pmol) representing the amount of fMet-tRNA bound in the
absence of IF3chl has been subtracted from each
value.
|
|
To test the effects from the short NH2- and the entire
COOH-terminal extension, IF3chlHT, consisting of the
homology domain surrounded by the tail and 15 residues of the head
(Fig. 1), was prepared and purified (Fig. 2, lane 8). The
induction of IF3chlHT resulted in a significant decrease in
cell growth and eventually appeared to cause cell lysis.
IF3chlHT had activity slightly lower than that of
IF3chlsrH but the same as that of IF3chlsHT and
IF3chlM when tested on E. coli 70 ribosomes with
poly(A,U,G) (Fig. 5A). When chloroplast 30 S subunits and
mRbcN were used, IF3chlHT had the same low
activity observed with IF3chlM (Fig. 5B). These
results indicate that IF3chlHT contains all the negative
regulatory elements present in IF3chlM and that the
negative effects due to sequences in the head and tail are
additive.
Basis for the Inhibitory Effect of Sequences in the Head and Tail
on the Activity of IF3chl--
In an attempt to understand
whether the low activity of IF3chlHT could be overcome by
raising the concentrations of 30 S subunits, mRNA, or IF2, assays
were carried out using different amounts of each component, separately.
As indicated in Fig. 6, increasing the
concentration of chloroplast 30 S subunits, mRbcN, or IF2 did not allow IF3chlHT to increase its activity relative to
the activity of IF3chlsrH. Similar results were obtained
when the levels of either IF2chl or E. coli IF2
were varied. These observations suggest that the low activity of
IF3chlHT is a complex phenomenon involving the interplay of
IF3chl with multiple components of the initiation
machinery.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of the concentrations of major assay
components on the inhibitory effects of head and tail sequences.
Different amounts of chloroplast 30 S ribosomal subunits (30 Schl), mRbcN, E. coli IF2
(IF2coli), or chloroplast IF2 (IF2chl )
were added in the chloroplast 30 S initiation complex formation assay.
Reaction mixtures contained 4 pmol of either IF3chlHT or
IF3chlsrH. The cross-hatched area represents the
activity obtained with IF3chlHT. The region between the
x-axis and the top of each open box represents the activity
obtained with IF3chlsrH.
|
|
The activities of several derivatives of IF3chl in
promoting initiation complex formation on chloroplast 30 S subunits
were tested in the presence of either the
or the
form of
IF2chl (data not shown) and E. coli IF2. The
results of this study indicated that all of the negative regulatory
effects from the head and tail regions are seen in the presence of
either form of IF2chl or E. coli IF2. The
natural mRNA used above (mRbcN) carries the initiation
region of the rbcL gene. This region does not have a
Shine/Dalgarno sequence. Indeed, about half of the chloroplast mRNAs in E. gracilis lack a Shine/Dalgarno sequence (35,
36). The negative effects of the head and tail were also tested with an
mRNA carrying the translational start site for the atpH
gene, which has a Shine/Dalgarno sequence just upstream of the start codon. The head and tail also inhibited the activity of the homology domain when this mRNA was used (data not shown).
Direct measurements of the abilities of various derivatives of
IF3chl to bind to chloroplast 30 S subunits were carried
out using sucrose density gradient centrifugation. For these
experiments, the appropriate derivatives of IF3chl were
incubated with chloroplast 30 S subunits. The bound factor was
separated from the free factor by sucrose gradient centrifugation. The
amount of IF3chl bound to the 30 S subunit was quantified
using an enzyme-linked immunosorbent assay. The amount of
IF3chl bound was calculated based on a standard curve
providing a measure of the response of each IF3chl
derivative to the antibody. The standard curves for each of the
derivatives are quite similar (Table I).
This observation was expected because the antibodies were raised
against the homology domain. The total amount of each derivative of
IF3chl bound to 30 S subunits and the estimated
Kobs are indicated in Table I. IF3srH had the
highest affinity for 30 S subunits, with a Kobs = 1.3 × 107 M
1. This value
is similar to the affinity of E. coli IF3 for E. coli 30 S subunits (K = 2.5 × 107 M
1) (37).
IF3chlerH and IF3chlsHT bound to 30 S subunits
with about 10-fold lower affinity than IF3chlsrH.
IF3chlHT showed the lowest ability to bind, with a
Kobs approximately 100-fold lower than that of
IF3chlsrH. These results suggest that the small
NH2-terminal extension region and the full tail interfere
with the ability of IF3chl to bind to 30 S subunits.
IF3chlHT, which contains both regions, showed the lowest
affinity for chloroplast 30 S subunits. Because the low activity of
IF3chlHT was not overcome by raising the concentration of
30 S subunits (Fig. 6), the head and tail must still down-regulate the
activity of IF3chl after this factor binds to 30 S
subunits.
View this table:
[in this window]
[in a new window]
|
Table I
Binding of IF-3chl to 30 S subunits
Approximate binding constants for the interaction of IF3chl
derivatives with chloroplast 30 S subunits were determined as described
under "Experimental Procedures." The 30 Schl-bound IF3 was
separated by sucrose gradient centrifugation. The amount of the
IF3chl bound to chloroplast 30 S subunits was quantified using the standard curve shown on the left.
|
|
Proofreading Ability of IF3chl and Its
Derivatives--
In E. coli, IF3 is believed to proofread
the selection of the initiator tRNA and the AUG start codon (10-12,
38). One procedure for monitoring this function is to examine the
ability of IF3 to destabilize preformed initiation complexes consisting
of 30 S ribosomal subunits carrying poly(U) and AcPhe-tRNA (33, 38). This destabilization affects all initiation complexes with the exception of those containing the initiator fMet-tRNA at an AUG codon,
which remains resistant to the destabilization induced by IF3 (39).
The ability of IF3chl to proofread in the chloroplast
system was examined by testing its ability to promote the dissociation of a preformed 30 S·poly(U)·AcPhe-tRNA complex.
IF3chlsrH has the greatest ability to destabilize the 30 S·poly(U)·AcPhe-tRNA complex (Fig.
7A). This observation is in
agreement with its greater ability to bind to 30 S subunits and to
promote initiation complex formation. IF3chlHT shows the
least activity in this assay, however, it still has some ability to
proofread. Surprisingly, all of the reduced proofreading ability seen
with IF3chlHT is also observed with IF3chlerH.
IF3chlsHT has more than half of the proofreading ability of
IF3chlsrH. These observations suggest that sequences in the
head region interfere with proofreading to a greater extent than those
in the tail region. The ability of derivatives of IF3chl to
discriminate between initiation complexes containing fMet-tRNA was also
tested (Fig. 7B). None of the derivatives that were examined destabilized the binding of fMet-tRNA to 30 S subunits. Indeed, some
stimulation of fMet-tRNA binding was observed with
IF3chlsrH even under the dilute conditions used in this
assay. This stimulation presumably reflects the high activity of this
derivative in promoting initiation complex formation.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Proofreading abilities of IF3chl
derivatives. A, the abilities of IF3chlsrH
( ), IF3chlerH ( ), IF3chlsHT ([itrio), and IF3chlHT ( ) to promote the dissociation of a
preformed 30 S·poly(U)·AcPhe-tRNA complex were analyzed in the
presence of the indicated amount of IF3chl as described
under "Experimental Procedures." The value for 100% complex
remaining is 1.0 pmol. B, the abilities of derivatives of
IF3chl to destabilize the initiation complexes containing
fMet-tRNA was tested under similar conditions. The value of
100% remaining bound represents 0.12 pmol.
|
|
 |
DISCUSSION |
IF3chl from E. gracilis is the first
organellar IF3 that has been cloned and over-expressed. The results
presented here indicate that a 9-residue sequence in the head region of
IF3chl and sequences in the tail play a negative regulatory
role in promoting initiation complex formation on chloroplast
ribosomes. Structural studies on E. coli and B. stearothermophilus IF3 (20-22) indicate that both factors fold
into two compact domains separated by a lysine-rich linker (Fig.
8). These two domains are formed by the
independent folding of sequences in the NH2-terminal and
COOH-terminal halves of the protein. The center of mass of the two
domains are separated by about 45 Å (22). The crystal structures of
the NH2-domain and COOH-domain of B. stearothermophilus IF3 (Fig. 8) indicate that both the
NH2 and COOH termini are oriented toward the central linker. The linker is highly basic and could play an important role in
interacting with 16S rRNA, tRNA, or mRNA (21). Both domains of IF3
are thought to be involved in ribosome binding (22). Because the head
and tail of IF3chl extend toward the linker region, they
may interact with specific residues in the linker region, which are
important for the binding of IF3 to the 30 S subunit and for its
function after it binds.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Structures of the NH2-terminal
and COOH-terminal domains of B. stearothermophilus IF3
taken from the x-ray coordinates. The two domains were
crystallized separately, and their exact orientation relative to one
another is not well understood. The centers of mass of the two domains
are about 45 Å apart (22). Both the NH2 and COOH termini
are oriented toward the central linker region.
|
|
The negative influence of sequences in the tail and of residues near
the junction between the head and the homology domain on the activity
of IF3chl suggests that there must be a mechanism by which
these effects may be modulated in vivo. One attractive hypothesis is that these regions down-regulate the intrinsic activity of IF3chl and that other factors in the chloroplast
alleviate this inhibition under appropriate conditions. This idea is
based on numerous observations that indicate that chloroplast protein synthesis is regulated in response to light and mRNA-specific trans-acting factors (40-47). Because IF3chl is required
for the translation of all mRNAs, it could play a key role in
modulating the activity of the chloroplast translational system as a
whole, for example, in response to light or developmental signals. In addition, trans-acting factors bound to specific chloroplast mRNAs could interact with IF3chl to recruit this factor for the
translation of a specific mRNA. The most logical region of
IF3chl to interact with such putative regulatory proteins
is the head. The rationale for this idea is as follows. The head has an
unusual amino acid sequence and, presumably, structure. It contains a
Pro-rich region reminiscent of many protein-protein interaction sites
and their flanking regions (48-54). Prominent examples of such sites
include proteins recognized by SH3 domains or the WW motif found in
many proteins participating in regulatory cascades. The
(GX)n motif (glycine-X motif, where
X indicates a large basic hydrophobic residue) following the
Pro-rich region would be expected to have significant structural
flexibility and could function as a flexible hinge region.
In a working model (Fig. 9),
IF3chl is visualized as being in a low activity state due
to the negative effects from the extensions on the homology domain
(Fig. 9). In this low activity state, the activity of
IF3chl would limit the rate of translation in the chloroplast to some basal amount. This level would, presumably, allow
the chloroplast to maintain the amounts of critical proteins at minimum
required levels. In the presence of appropriate environmental signals
(for example, in conditions promoting photosynthesis), a regulatory
factor interacts with the head on IF3chl relieving the
inhibitory effects and allowing the homology domain to become fully
active. A protein affecting the activity of IF3chl could potentially act either in general, increasing the overall rate of
chloroplast protein synthesis, or more specifically, promoting the
translation of specific mRNAs. In the latter case,
IF3chl can be envisioned as playing a role in tying
mRNA-specific trans-acting factors to the general translational
machinery. Current efforts are designed to gain insight into the
factors that modulate the activity of IF3chl and, thus, the
rate of chloroplast protein synthesis.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
Model for the regulation of
IF3chl activity by interaction of proteins that bind to
sequences in the head region.
|
|