Nov 3, 1993 - Qiong L i d , Lan Ma$, William Burkharts, and Linda L. SpremulliSnll ...... Robson algorithms predict that this region is largely a-helical. Thus, the ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Val . 269,No.
Issue of April 1, pp. 9436-9444, 1994 Printed in U.S.A.
Isolation and Characterizationof cDNA Clones for Chloroplast from Euglena g r a d i s * Translational Initiation Factor-3 (Received for publication, November 3, 1993)
Qiong L i d , Lan Ma$, WilliamBurkharts, and Linda L. SpremulliSnll From the $Department of Chemistry a n d the YLineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill, North Carolina 27599 a n d the $Department of Structural andBiophysical Chemistry, Glaxo Research Institute, Research Diangle Park, North Carolina 27709
A complete cDNA clone encoding Euglena gracilis of 30 S subunits for initiation complex formation. Second, IF-3 chloroplast translationalinitiation factor 3 (IF-3,,J has promotes the conversion of a complex referred toas the preterbeen obtained. Analysis ofthe sequence indicates that nary complex to the ternary complex (7, 8). The preternary the IF-3,,, mRNAcontainsthe spliced leader foundat the complex contains both the mRNA and the Net-tRNA the on 30 5’ end of nuclear encoded mRNAs in E. gracilis. The S subunit, but they are not yet hydrogen-bonded. In the ternary IF-3,,, encodes a 537-amino acid complex, the mRNA and Net-tRNA interact stably through open reading frame for protein. IF-3,, appears to be divided into four domains. codon:anticodon hydrogen bonding.IF-3 also playsa proofreadThe first 140 amino acids correspond to a transit peptide ing role in initiation by promoting the selection of the initiator required for the import of IF-3,,, into the chloroplast. tRNA as opposed to elongator tRNAs in the 30 S initiation The mature form of IF-3,,, encompasses domains 2-4 and complex (9-11). No factor directly comparable to IF-3 is found is about twice the size of Escherichia coli IF-3. The secin the eukaryotic cytoplasmic protein biosynthetic system. ond domainhas no homologyto other known proteins. It IF-3,,, can replace E. coli IF-3 in promoting 70 S initiation begins with astretch of 35 residues, of which about30% are proline. Downstream from this region is a stretch of complex formation on E. coli ribosomes. This observation sugabout 25 amino acids with a repeating ( G X ) , motif fol- gests that the chloroplast factorhas retained many of t h e fealowed by a very acidic region. The third domain com- tures and functions of the corresponding prokaryotic factors. 31 However, IF-3,,, is physically unusual compared to the bacteprises a region of about 175 residues and has between and 37% homology to the IF-3s found in other organisms.rial factor.E. coli IF-3 is a monomeric protein witha molecular three forms The IF-3 homology domain is followed by an acidic re- mass of 22 kDa. IF-3,,, has been resolved into gion which has nodetectablehomology to other se- designated a , p, a n d y. All of these forms of IF-3,,, function as quences. Analysis of E. gracilis genomic DNA suggests monomers but are about twice the size of the corresponding that there are about four copies of the IF-3,,, gene, one of prokaryotic factors. E. coli IF-3 is a basic protein, which has a which is probably a pseudogene. The activity of IF-3,,, is strong RNA binding activity (12). It binds tothe 30 S subunit by inducible by light. However, theIF-3,,, mRNA is present a specific interaction with the16 S rRNA. In contrast, IF-3,,, i s in approximately equal amounts in both dark- and light- an acidic protein, which does not have a strong RNA binding grown cells, suggesting that the light-dependent induc- activity. tion of IF-3,,, activity is post-transcriptional. Both IF-2,,, a n d IF-3,,, are the products of nuclear genesin E. gracilis. In addition,the activities of both proteins are induced Two chloroplast translational initiation factors (IF-2,,, a n d 1F-3chl)1 have been purified and characterized from Euglena gracilis (1-3). IF-2,,, promotes the binding of Net-tRNA to chloroplast 30S ribosomal subunits. It is a large complex factor ranging in size from 200 to 700 kDa. The active of forms IF-2,,, comoccur as dimeric,tetrameric,andprobablyhexameric plexes of 97-200-kDa subunits. This factor has little activity binding Net-tRNA to the Escherichia coli ribosomes. IF-3,,, enhances the poly(A,U,G)-dependent binding of the initiator tRNA to chloroplast 30 S subunits (3, 4). In E. coli IF-3 is ( 5 , 6). thought to play several roles in the initiation process First, it binds to the 30 S subunit and inhibits its association with the 50 S subunit. In thisway, IF-3 helps ensure a supply
by exposure of the cells to light (131, as is the development of the chloroplast itself. Chloroplast proteins encodedby nuclear as precursors genes are generally synthesized in the cytoplasm that have extensions at the N terminus. The chloroplastsof E. gracilis are surroundedby three membranes as opposed to the two plastid membranes observed in higher plants and in many algae. The transit peptidefor chloroplast proteinsin this orgain nism must, therefore, contain the information required to direct the precursor polypeptide through all three of these membranes.
EXPERIMENTALPROCEDURES Materials-Oligonucleotide primers weremade in the Lineberger Comprehensive Cancer Center at the University of North Carolina. [CP~~PI~CTP (3000 Ci/mmol) was obtained fromDuPontNEN. Pure nitrocellulose blotting membranes were obtained fromSchleicher & *This work has been supported in part by National Institutes of Schuell, and Zeta-Probe Blotting Membranes were purchased from BioHealth Grant GM24963. The costs of publication of this article were Rad. Restriction enzymes werepurchased from U.s.Biochemical Corp., defrayed in part by the payment of page charges. This article must Pharmacia, and Life Technologies, Inc. Oligo(dT)-cellulosem e 3 was therefore be hereby marked “aduertisement”in accordance with 18 obtained from Collaborative Research. Sequenase@version 2 DNA seU.S.C. Section 1734 solely to indicate this fact. of the sequence The nucleotide sequence($ reportedin this paper has been submitted quencing kit was from U.S. Biochemical Corp. Analysis to the GenBankTMIEMBL Data Bank with accession number(s) L23760. was done with the Genetics Computer Group (GCG) sequenceanalysis programs (version 7.2, October, 1992) running on the University of 11 To whom correspondence shouldbe addressed. The abbreviations used are: IF, initiation factor; bp, base pairb); North Carolina VAX computer. Light Induction of Chloroplast Development in E. gracilis-The kbp, kilobase pair(s); kb, kilobase(s);PCR, polymerase chain reaction; Tp,annealing temperature; SL, spliced leader; ER, endoplasmicreticu- growth of E. gracilis Klebs var. bacillaris Cori (E. gracilis B) and the light induction of chloroplast development werecarried out basically as lum.
9436
Chloroplast Initiation Factor 3
9437
Screening cDNA Libraries-Approximately 1.5 x lo6 plaques from described (13). Cellsin 50 ml of media were grown in the dark t o a cell the random-primed cDNA library and approximately 250,000 plaques density of 2 x lo6 celldml. The cultures were transferred to200 ml of from the oligo(dT)-primed library were plated on E. coli XL1-Blue and resting media and kept in the dark for 3 more days in order to exhaust a s described (21). Appropriate the carbon source. The cells were then either maintained in the darkplaque or hybridization was carried out exposed to light for 72 h. Chloroplast development was monitored by probes were labeled using the random priming method (22). Positive purified. The determining the chlorophyll content per cell (14). Cells (0.32-0.58 g) plaqueswereselected,replatedandrescreeneduntil were harvested by centrifugation, and RNA was extracted according to pBluescript SK(-) phagemid clones containing positive inserts were excised from the Lambda ZAP I1 and selected according t o the manuthe procedure described (15). Purification of IF-3,,, and Peptide Sequence AnaZysis--IF-3,,, p (mo- facturer's instructions (Stratagene). DNA Sequencing-All the putative clones were sequenced by the lecular mass 42 kDa) was purified as described (3), except that the TSKgel heparin-5PW column was omitted. After the TSKgel DEAE- dideoxynucleotide chain termination method (23) and subjected to au5PW column, about 30 pg of pure IF-3,,, p was obtained for partial tosequencing in theUniversity of North Carolina DNA Sequencing Facility. M13 universal and reverse primers,as well as a number of synamino acid sequence analysis. For this procedure, a sample of IF-3,,, p derived from the IF-3,,, was applied to a Hewlett-Packard hydrophobic sequencing column ac- thetic oligodeoxyribonucleotide primers sequence were used. Each region was sequenced completely in both cording to the manufacturer's protocol. The protein was reduced and alkylated in situ by dripping a buffer containing 20 m dithiothreitol, directions. Northern and Southern Blots-RNA was analyzed on 0.9% agarose 50 m 4-vinylpyridine in 6 M guanidine hydrochloride containing0.5 M Tris-HC1, pH 8.6, and 5 m~ EDTA through the column a t room tem- gels in the presence of 1 M glyoxal and 50% dimethyl sulfoxide as perature for 15 min. In situ proteolytic digestion with endoproteinase described (24). Total genomic DNA was prepared essentially as deLys-C (Wako Chemicals) was performed according to Burkhart (16). In scribed (251, and 20 pg were digested with 200-300 units of the indicated restriction enzyme. Digests were run on a 0.7% agarose gel at 25 situCNBrcleavagewasperformedon a Hewlett-PackardProtein Chemistry Stationaccording to the manufacturer'sprotocol using a 6-h V for 17 h. RNA and DNA were transferred from the agarose gels to cleavagetime.Afterdigestion,peptideswereseparated by reverse Zeta-Probe blotting membranes as recommended by the manufacturer. phase high performance liquid chromatography usinga Hypersil ODS For hybridization reactionsthe blotted membranes were incubated with column (2.1x 100 mm; Hewlett-Packard) employing a linear gradient of 2 x lo7 cpm of 32P-labeledprobe (2 x lo9 c p d p g ) i n 1mM EDTA, 0.5 M 8 4 8 % acetonitrile in 0.1% trifluoroacetic acid over 80 min with a flow NaH,PO,, pH 7.2, and 7% SDS at 65 "C for 1P18 h. The membranes rate of 100 pVmin. Peptides were sequenced on theApplied Biosystem were then washed using the standardprotocol described by the manumodel 477A Protein Sequencer with themodel 120A PTH Analyzer. facturer. For reuseof the blot, it was stripedby incubation in 0.1x SSC (0.015 M NaCl,O.0015 M trisodium citrate) and 0.5%SDS at 95 "C for 30 RNA Isolation and Construction of cDNA Libraries-Total RNA was extracted from light-grown E . gracilis essentially as described (17). min. This washing procedure was then repeated prior to reprobing the DNA was removed by lithium chloride precipitation.Poly(AY RNAwas blot. purified by oligo(dT1-cellulosechromatography (18).First strandcDNA was synthesizedby reverse transcription of 5 pg of poly(AY RNA using RESULTS AND DISCUSSION either random hexamers oroligo(dT),,,, a s primers and Moloney muIsolation of cDNA Clonesfor IF-3c,,l-IF-3chl is theproduct of rine leukemia virus reverse transcriptase (Pharmacia). A SephacrylR S-400 spun column was used to select cDNAs over 400 bp. Second nuclear gene in E. gracilis (13). In orderto obtain cDNAclones strand cDNA was synthesized using RNase H nicking, followed by DNA of this factor, it was necessary to obtain partial peptide sesynthesis using E . coli DNA polymerase I as suggested by Stratagene. quence information on this protein. Unfortunately, purified IFEcoRYNotI adaptors were ligated to the double-stranded cDNAby blunt 3,,, is an extremely sticky protein and is easily lost by adsorpend ligation using T4 DNA ligase. The cDNA was ligated to EcoRItion onto surfaceswhen present at the low concentrations digested A ZAP I1 (Stratagene) and packaged with A phage packaging resulting from its purification. Techniques commonly used to extract (Stratagene). The random-primed libraryyielded a total of 3 x concentrate and change the buffer of a protein sample lead to lo6independent recombinants, while the oligo(dT)-library contained 5x lo4 independent plaques. The latter library was amplified in E. coli significant losses ofIF-3,,, even when siliconized tubes were XL1-Blue resulting a library containing 1 x 10, plaque-forming units/ used. In order to minimize loses during sequence analysis, a ml. method for the in situ digestion of IF-3,,, was used (16). With Preparation of Specifically Primed cDNA-A first strand cDNA was prepared by priming 5 pg of E. gracilis poly(A)+RNA with 60 pmol of this approach peptides derived from IF-3,,, were obtained folLys-C or CNBr. Eight Primer P4 (see Table 11) and 1200 units of Moloney murine leukemia lowing digestion with endoproteinase virus reverse transcriptase basically as described (19). Aliquots corre- peptides ranging from 6 to 34 amino acids in length were obsponding to 1/10 of the cDNA synthesis reaction were used in the PCR tained from endoproteinase Lys-C cleavage, and five peptides amplification reactions described below. were obtained from the CNBr digestion (Table I). Two sets of PCRAmplification-PCR amplifications were performed in a Perkinpeptides overlapped allowing the deduction of two longer Elmer DNA Thermal Cycler. All the primers used carried a convenient restriction siteat their5' end (see Table11). Reaction mixtures (100p1) stretches of amino acid sequence (peptideA has 54 amino acids, contained 60 pmol of each primer, 2.5 units of Taq DNA polymerase and peptide B has 32 amino acids; Table I). No sequence infor(Promega), 0.2m~ dNTPs, the buffer system recommendedby Perkinmation could be obtained directly from the N terminus, sugElmer, and either 0.1 of pggenomic DNA or an aliquot of the specifically gesting that it might be blocked. Several of the peptide seprimed cDNA prepared as described above. The template DNA was quences obtainedshowed a significant percentageof amino acid denatured by incubation for 5 min at 94 "C followed by 5 min at 85 "C prior to the addition of Taq DNA polymerase. Amplification reactions sequence identity to E. coli IF-3 (Table I), allowing the predicwere performed using a n annealing temperature (T,) calculated as de- tion of their positions relative to each other. Other peptides, scribed (20). Duringthe first threecycles of PCR, the T,of the primer however, showed little or no sequence identity with the prowithout the added restriction site was used. The remaining 35 cycles karyotic factors.This observation is not surprising, since IF-3,,, used the T, of the primer including the added restriction site. For is about twice the size of E. coli IF-3 and, thus, wouldbe primer combinationsthat differed in T,,the lower value was used in theexpected to have significant regions of sequence with little rereaction. During each round of PCR, denaturation of the DNA was done semblance to the bacterial factor. a t 94 "C for 1min, primer annealing was at the specified T,for 1.5 min, Peptides A and B exhibited homology to their prokaryotic and polymerization was carried out at 72 "C for 2 min. In the last cycle, formed the basisof the initial stratthe reaction time at 72 "C was extended to 10 min toallow completion counterparts, and their use of chains and the incubations were thencooled to 4 "C. egy designed to obtaina portionof the IF-3,,, gene. Based on its PCR reactions involving a second round of amplification using dif- sequence, peptide A was predicted to be derived from a region ferent primers were prepareda s described above, except that theycon- of IF-3,,, corresponding to a sequence near theN terminus of E. tained 1pl of the reaction mixture obtained from the first round of PCR. The reaction mixtures were analyzedon 1.4% agarose or 3% Nusieve@ coli and Bacillus stearothermophilus IF-3 (Fig. lA). Peptide B GTG-agarose gels. Specific bands were identifiedby ethidium bromide was predicted to be on the C-terminal side of peptide A. Four staining and eluted from the gel using the Geneclean or Mermaid kitsdegenerate oligonucleotide primers for PCR were designed from BiolOl. based on these peptide sequences and on the codon usage of E.
9438
Initiation Chloroplast
Factor 3
TABLEI Amino acid sequences of IF-3,,, peptides* Sequences underlined were usedfor designing degenerate PCR primers. The designationX represents an ambiguous residue. Homology to E. coli IF-3
Amino acid sequence
Lys C peptides 1. LGVMSRQEALELAEAEDIDLVLVSIDTDPPVa 2. RFQNDLADMGK~ 3. YPQLXLVXAVPGGRDEK 4. VSMEFK~ 5. GSRSSTLSNLGMGLGLGLGXGXGFGXGFGXGRGF 6. VIAERRAERDRK~ 7a. IGQHDYDVRVKQA~
7b.
FLEGGHRIK~
8.
GRENQFVEIGRAVMK~
Position
% Identity
34-65 147-157
51 30
-
-
-
-
117-125 132-146
40
CNBr peptides 9. MGLGLGLGKGXGFGXGFGLGXGFPVEEEVEEE~
-
10. MGKADAVPXKLXTFXILNXAPA
128-151 15-36 37-64
11. MEFKGRENQFVEIGRAVMKRFQND~ 12. MNEDIKYPQLRLVRAVPGGRDEKLGVa 13. MSRQEALELAEAEDIDLVLVSIDTDP' a
55
33 36 53
These peptides overlap into peptide A MNEDIKYPQLRLVRAVPGGRDEKLGVMSRQEALELAEAEDIDLVLVSIDTDPPV.
* These peptides overlap into peptideB: VSMEFKGRENQFVEIGRAVMKRFQNDLADMGK.
e
Indicates the % identity was
