Regulation of the Activity of Chloroplast Translational Initiation Factor 3 by NH2- and COOH-Terminal Extensions*

Nan-Jun YuDagger and Linda L. SpremulliDagger §

From the Dagger  Department of Chemistry and § Lineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill, North Carolina 27599-3290

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The mature form of the chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis consists of an internal region homologous to prokaryotic IF3 flanked by long NH2- and COOH-terminal extensions. Sequences in these extensions reduce the activity of the homology domain in promoting initiation complex formation with chloroplast mRNAs and 30 S ribosomal subunits. A series of deletions of the NH2- and COOH-terminal extensions of IF3chl were constructed and tested for their effects on the activity of the homology domain. About half of the inhibitory effect arises from sequences within 9 residues of the junction between the NH2-terminal extension and the homology domain. The remaining inhibitory effect is the result of sequences in the COOH-terminal extension. The equilibrium constant governing the binding of the homology domain of IF3chl to 30 S subunits is estimated to be 1.3 × 107 M-1. Sequences close to the junction of the NH2-terminal extension and the homology domain reduce this binding constant about 10-fold. Sequences in the COOH-terminal extension have a similar negative effect. The negative effects of these two regions are cumulative, resulting in a 100-fold reduction of the binding constant. The 9 residues at the NH2-terminal extension effectively prevent the proofreading activity of IF3chl. The entire COOH-terminal extension reduces the proofreading ability by about half. These results are discussed in terms of the proposed three-dimensional structure of the homology domain of IF3chl.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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, alpha , beta , and gamma . The alpha  form has a molecular mass of about 34 kDa, whereas the beta  and gamma  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
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Abstract
Introduction
Procedures
Results
Discussion
References

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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.


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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).


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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.


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Fig. 3.   Inhibitory effects of the head and tail regions on the activity of the homology domain of IF3chl. A, activities of IF3chlsrH (open circle ), IF3chlHdH (bullet ), IF3chlsHT (down-triangle), and IF3chlM (black-down-triangle ) 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).


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Fig. 4.   Small regions of the head are sufficient to confer the full inhibitory effect of the head. A, activities of IF3chlerH (bullet ) and IF3chlrH (down-triangle) compared with IF3chlHdH (open circle ) and IF3chlsrH (black-down-triangle ) 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 <FR><NU>1</NU><DE>3</DE></FR> 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 <FR><NU>2</NU><DE>3</DE></FR> of the tail region are essential for the inhibitory effect of the tail.


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Fig. 5.   Effect of the tail on IF3chlsrH and additive effects of sequences in the head and tail. A, activity of IF3chlsHT/3 (open circle ) and IF3chlHT (square ) compared with IF3chlsHT (down-triangle), IF3chlsrH (bullet ), and IF3chlM (black-down-triangle ) 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.


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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 IF2alpha (IF2chlalpha ) 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 alpha  or the beta  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.

                              
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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.


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Fig. 7.   Proofreading abilities of IF3chl derivatives. A, the abilities of IF3chlsrH (black-down-triangle ), IF3chlerH (bullet ), IF3chlsHT ([itrio), and IF3chlHT (open circle ) 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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.


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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.


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Fig. 9.   Model for the regulation of IF3chl activity by interaction of proteins that bind to sequences in the head region.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM24963.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: Dept. of Chemistry CB# 3290, University of North Carolina, Chapel Hill, NC 27599-3290. Tel.: 919-966-1567; Fax: 919-966-3675; E-mail: Linda_Spremulli{at}unc.edu

1 The abbreviations used are: IF, initiation factor; IF3chl, chloroplast translational initiation factor 3; IF3chlM, mature form of IF3chl; Hd, head; H, homology; T, tail.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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