M.capricolum. INTRODUCTION. UAA, UAG and UGA are termination codons in the universal genetic code. In eubacteria such as Escherichia coli (1,2) and.
Nucleic Acids Research, 1993, Vol. 21, No. 6 1335-1338
Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum Yuji Inagaki*, Yoshitaka Bessho and Syozo Osawa+ Laboratory of Molecular Genetics, Department of Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan Received January 29, 1993; Accepted February 18, 1993
ABSTRACT In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coil S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.
To clarify this, a cell-free system of M. capricolum was constructed for translation of the added synthetic mRNAs containing UAA, UAG or UGA in-frame. When tryptophan was removed from the system, translation ceased just before UGA and the bulk of the synthesized peptides was attached to the ribosomes. The peptides were released upon incubation with puromycin or the E.coli or B.subtilis S-100 fraction. These results indicate that UGA-dependent RF-activity is absent or at least very weak.
MATERIALS AND METHODS
INTRODUCTION
Cell culture Mycoplasma capricolum [American Type Culture Collection 27343 (Kid)] cells were grown as described (10) and stored at -0°C until use. Escherichia coli K12 JCH1 11 (F-, met-, trp-) and Bacillus subtilis BD170 (thr-, trp-) cells were grown in the LB medium until middle log phase at 37°C with shaking. Cells were collected by centrifugation at 8,000xg at 0°C and washed twice with M9 medium (0.6% Na2HPO4/0.3 % KH2PO4/0.5 % NaCl/0. 1% NH4Cl/2mM MgSO4 /O.lmM CaCl2/0.2% glucose). E.coli or B.subtilis cells were subjected to tryptophan starvation by shaking in M9 medium including methionine (100/lg/ml) or threonine (10Og/ml) at 37°C for 3 hrs, collected and stored at -70°C until use.
UAA, UAG and UGA are termination codons in the universal genetic code. In eubacteria such as Escherichia coli (1,2) and Bacillus subtilis (3), two protein factors, release factor 1 (RF-1) and release factor 2 (RF-2), participate in the recognition of the termination codons and in release of nascent polypeptides from the ribosomes. RF-1 recognizes UAA and UAG, whereas RF-2 recognizes UAA and UGA (1). It is well established that a bacterium M. capricolum uses UGA as a tryptophan codon instead of termination signal (4-7). Codon UGA is translated by tRNATUA. It is then expected that M. capricolum does not possess the UGA-dependent RF-activity in response to the use of UGA as a regular tryptophan codon.
Preparation of the S-30 fraction and S-100 fraction The M. capricolum S-30 fraction was prepared as described (8), and was dialyzed against 2 1 of buffer A (1OmM Tris-HCl pH7.8/lOmM magnesium acetate/60mM NH4CI), with changes at 1 hr-interval for 6 hrs to remove amino acids and stored at -70°C until use. The E.coli and B.subtilis S-100 fractions were prepared as described (9), dialyzed against 2 1 of buffer A with changes at 1 hr-interval for 8 hrs and stored at -70°C until use. The final protein concentration of the S-30 fraction was 9.8mg/ml, and those of the E. coli and B.subtilis S-100 fraction were 9.0mg/ml and 3.0mg/ml, respectively.
To whom correspondence should be addressed 'Present address: Cosmo Annex ID, 7 Umezono, Osaka 569, Japan *
Hiroji-cho, Showa-ku, Nagoya 466, Japan. After June 1993: Biohistory Research Hall, Takatsuki-shi,
1336 Nucleic Acids Research, 1993, Vol. 21, No. 6 Preparation of synthetic messenger RNAs The fragments having sequences corresponding to mRNAs with restriction enzyme sites (Pst I and Sac I) for cloning were synthesized by Gene Assembler Plus (Pharmacia LKB) (Fig. 1). The synthesized inserts were ligated to plasmid vector Bluescript II KS+ (stratagene) at the Pst I and Sac I sites. The plasmids were transformed to E.coli DHIOB. The plasmids carrying correct insert were selected by DNA sequencing. The transcription of mRNA from DNA with the T7 RNA polymerase was done as described (10). All enzymes used were purchased from Takara Shuzo Co., LTD.
Cell-free translation The standard reaction mixture contained, in 90p1, 50mM Tris-Cl pH7.8, 3.75mM of magnesium acetate, 60rnM of NH4Cl, lmM of dithiotheitol, 5i4M of each of methionine and tryptophan, 666KBq of [3H]isoleucine (3.74TBq/mmol, Amersham), 7,ug of synthetic mRNA and 27,ad of the S-30 fraction. In some experiments, 16t1 of the M. capricolum S-30 fraction were used with addition of 16yd the B.subtilis S-100 fraction or 5,1i of the E.coli S-100 fraction. The reaction mixture was incubated at 37°C. Ten gl aliquot was taken out at intervals and was treated with 1N NaOH at 370C for 15 min, followed by 20% (wt/vol) trichloroacetic acid (TCA) treatment at 0°C for 30 min. The TCA-insoluble material was collected on glass filters (Whatman GF/C) and thoroughly washed with cold 10% TCA. The radioactivity was measured by liquid scintillation counter (Tri-Carb).
Sucrose-gradient centrifugation Ninety ,ul of the reaction mixture were incubated at 37°C for 10 min. When lmM puromycin was added, the mixture was further incubated at 370C for 10 min. It was then layered on 27 ml of 5-20% (wt/vol) sucrose-gradient made in the translation buffer (5OmM Tris - HCI pH7.8/3.75mM magnesium acetate/60mM NH4Cl), and centrifuged in Beckman SW-25.1 rotor at 35,00Oxg for 13 hrs at 0°C. Fractions were collected from the bottom of the tube and the alkali-stable and TCA-insoluble radioactivity in each fraction was measured. mRNA(UAA)
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RESULTS
Design of synthetic messenger RNAs The synthetic mRNAs used in this study are shown in Fig. 1. A pair of codons to be tested, UAA, UAG or UGA, was preceded by ten AUU isoleucine codons and followed by two UAA termination codons.
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Figure 2. Incorporation of [3H]isoleucine into alkali-stable and TCA-insoluble materials in a cell-free translation of synthetic mRNAs. The codon to be tested (test codon), UAA, UAG or UGA, in the mRNA is shown by triplet. '+Trp' or '-Trp' indicates the translation in the presence or absence of tryptophan. -mRNA: mRNA was not added.
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Figure 1. Sequence alignment of three mRNAs, mRNA(UAA), mRNA(UAG) and mRNA(UGA). The synthetic mRNAs include, 5' to 3' end, the Shine-Dalgamo sequences (indicated as S.D.), an initiation methionine codon AUG (M), ten AUU isoleucine codons (I), two test codons and two UAA termination codons (*). Codons to be tested are UAA (*), UAG (*) and UGA (W). Sequences shown by large letters are transcripts from insert DNAs; those by small letters are from plasmid vector sequences. Regions from Sac I to Pst I were used as the restriction enzyme sites for cloning. The Hind Em site was used for linearization of plasmid and used as template.
10
Figure 3. Sucrose-gradient centrifugation of reaction mixture labelled with [3H]isoleucie. (A) Translation of mRNA(UAA). (B) Translation of mRNA(UAG). (C) Translation of mRNA(UGA) in the presence of tryptophan. (D) The same as (C) but in the absence of tsyptophan. (E) Translation of mRNA(UGA) followed by incubation with puromycin.
Nucleic Acids Research, 1993, Vol. 21, No. 6 1337
Cell-free translation Translation of the synthetic mRNAs was measured by [3H]isoleucine as the labelled amino acid in the presence or absence of tryptophan. With all mRNAs [mRNA(UAA), mRNA(UAG) and mRNA(UGA)], [3H]isoleucine was incorporated into TCA-insoluble materials with nearly equal efficiency (Fig. 2). No incorporation occurred in the absence of mRNA. Sucrose-gradient centrifugation of reaction mixture labelled with [3H]isoleucine To investigate the state of the [3H]isoleucine-labelled peptides, the incubated reaction mixture was examined by sucrose-gradient centrifugation. In the case of mRNA(UAA) or mRNA(UAG), the bulk of the synthesized peptide was recovered in the solublefraction (top of the gradient; Figs. 3A and B), indicating that the peptides synthesized were released from the ribosomes at UAA or UAG after translation of AUU isoleucine codons (Figs. 1 and 4A). With mRNA(UGA) in the presence of tryptophan, the peptides were released from the ribosomes (Fig. 3C), indicating that codon UGA was translated as tryptophan and the peptide-chain termination occurred at UAA termination codon following UGA (Figs. 1 and 4B). Some [3H]radioactivities in the ribosome region in Figs. 3A, B, C would most probably represent the unfmished [3H]isoleucine peptides in the course of translation. When tryptophan was absent, the bulk of the peptides was detected on the 70S ribosome fraction with some in the soluble-fraction (Fig. 3D), indicating that the translation mostly
ceased at UGA without an appreciable release of the peptides (Figs. 1 and 4C). Puromycin released the [3H]isoleucinelabelled peptides from the ribosomes completely (Fig. 3E).
Analyses with heterogeneous cell-free translation system Since RF-2 of Escherichia coli or Bacillus subtilis recognizes tenmination codons UAA and UGA (1, 3), the E. coli or B.subtilis S-100 fraction was added to the M. capricolum S-30 fraction in the presence of mRNA(UGA) and in the absence of tryptophan. [3H]isoleucine was incorporated into alkali-stable and TCA-insoluble materials (Fig. 5), which were almost exclusively recovered in the soluble-fraction after sucrose-gradient centrifugation (Figs. 6A and B). No [3H]isoleucine incorporation occurred in the absence of the M. capricolum S-30 fraction and in the presence of the E.coli or B.subtilis S-100 fraction (Fig. 5), indicating the absence of the ribosomes in the S-100 fraction added.
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B:
mRNA(UGA) +Trp
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Figure 5. Incorporation of [3H]isoleucine into aLkali-stable and TCA-insoluble materials with mRNA(UGA) in the presence of the E.coli and B.subtilis S-100 fraction and in the absence of tryptophan. Translation of mRNA(UGA) in the presence of the M.capricol4m S-30 fraction and the E.coli (A) or B.subtilis (]) S-100 fraction. Translation with the M.capricolum S-30 fraction only (0). Translation in the absence of the M. capricolum S-30 fraction but in the presence of the Ecoli and B.subtilis S-100 fraction (0).
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