J. Biochem. 119, 811-816 (1996) Characterization of a B. subtilis Minor Isoleucine tRNA Deduced from tDNA Having a Methionine Anticodon CAT1 Jitsuhiro Matsugi,2 Katsutoshi Murao, and Hisayuki Ishikura Laboratory of Chemistry, Jichi Medical School, 3311-1 Minamikawachi-machi, Tochigi 329-04 Received for publication, December 1, 1995 Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine. We isolated this tRNA and determined its sequence including modified nucleotides. Modified nucleotide analyses using TLC, UV, and FAB mass spectros copy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N6 - lysino)-1-ƒÀ-D-ribofuranosyl pyrimidine]. As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA. Key words: Bacillus subtilis, FAB mass spectroscopy, lysidine, modified nucleotide, tRNA. In the codon table, isoleucine is assigned to three codons, lysidine in the wobble position can form a base pair only AUU, AUC, and AUA, but theoretically two kinds of with adenosine, not with guanosine, and consequently the anticodon (GAU and UAU) are sufficient to decode these minor tRNA11e can read only the AUA codon. At the same three codons. To decode the AUA codon, some organisms time, lysidine contributes to a change in charging specificity utilize a minor species of tRNA11e whose first letter of the from methionine to isoleucine (7). anticodon is a unique or an unknown modified nucleotide. In B. subtilis, as described above, the structure of the For example, in Escherichia coli, a Gram-negative eubac tRNA transcribed from tDNA(CAT) remained unknown. teria, and Mycoplasma capricolum, a derivative of a Gram Since we were studying the tRNA structure and function of positive eubacteria, it has been reported that their minor B. subtilis (8, 9), this prompted us to attempt to determine tRNA1es have lysidine [4-amino 2-(N6-lysino)-1-ƒÀ-D-ribo the RNA sequence including its modification pattern. In this furanosyl pyrimidine; referred to as L in this paper], which paper, we report the structure of the tRNA11e transcribed is posttranscriptionally converted from cytidine (1, 2). from tDNA(CAT). Some plant mitochondria and chloroplasts, and phage T4 have an unknown modified nucleotide possibly converted MATERIALS AND METHODS from cytidine (3-5). Bacillus subtilis is a Gram-positive eubacteria, and its Isolation and Purification of the Minor Isoleucine modification patterns used in the codon-anticodon relation tRNA„ŸB. subtilis W168 unfractionated tRNA was prepar ship show some differences from those of Gram-negative ed as described previously (10). The bulk tRNA (134,500 bacteria such as E. coli. In addition, B. subtilis is known to A260 unit) was purified successively by DEAE Sepharose have characteristic tRNA gene clusters, Green and Vold CL-6B, Sepharose 4B, and BD cellulose column chromato have reported that trnB, one of the tRNA gene clusters, graphies. For the fractionation, measurement of [14C] iso encodes 21 tRNAs (6). Analysis of their sequences revealed leucine acceptor activity using E. coli aminoacyl-tRNA that 2 of them are isoleucine tDNAs. One is a tDNA1e with synthetase (11) and the dot blot hybridization methods (see the GAT anticodon and the other is one with the CAT below) were employed. The final purification was done by anticodon [referred to tDNA(GAT) and tDNA(CAT) re semipreparative HPLC (hydroxyapatite column, 21.4•~ spectively]. Comparison with other known tRNA1es sug 100mm, TAPS, TONEN, buffer A; 5mM sodium phos gested that the tDNA(CAT) accepts isoleucine, though its phate, pH 8.0, buffer B; 300mM sodium phosphate, pH anticodon sequence corresponds to methionine. Thus, if 8.0) (12) and 360 A260 of the tRNA was obtained. there is no change in its anticodon, competition with the Dot Blot Hybridization-A DNA probe for the dot blot methionine codon by tRNA Met and tRNA11e will occur and hybridization was prepared using the phosphoramidite lead to mistranslation. Therefore, a mechanism such as method (13). [ƒÁ-32P]ATP (3,000Ci/mmol) was purchased posttranscriptional modification targeting the anticodon, from Amersham. The hybridization was done by the which is also one of the strong identity sites, must be procedure of Yokogawa et al. with several modifications required to change the codon recognition. In fact, in E. coli, (14). The tRNA solution (2ƒÊl) was directly spotted onto a nylon membrane (Hybond-N, Amersham) and dried. The 1 The nucleotide sequence data of a B. subtilis minor tRNA11ereported membrane was irradiated by UV light at 254nm for 5min in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D26446. and preincubated in 5•~Denhardt's solution, 5•~SSPE (20•~ 2 To whom correspondence should be addressed . Phone: +81-285-44 SSPE contains 3M NaCl, 0.2M sodium phosphate, pH 7.4, 2111 (Ext. 3366), Fax: +81-285-44-2858 and 20mM Na2EDTA) and 0.5% SDS at 60•Ž for 1h. The Vol. 119, No. 4, 1996 811 812 J. Matsugi et al. membrane was then incubated in new hybridization buffer around this region. The 5•L-terminus of the probe was (same composition as the prehybridization solution) con labeled for the hybridization by [ƒÁ-32P] ATP and polynu taining 5•L-[32P]-labeled probe (obtained from the kination cleotide kinase. of 100 pmol oligonucleotide with [ ƒÁ-32P] ATP) at 37•Ž By large-scale cultivation of B. subtilis, we obtained overnight. After the hybridization, the membrane was 134,500 A260 of the unfractionated tRNA. To isolate the washed five times with 3•~SSC (450mM NaCl, 45mM tRNA11e from the bulk tRNA, several column chromatogra sodium citrate pH 7.0) at room temperature. The mem phies were employed. As an example of one of the steps, the brane was autoradiographed using X-ray film with an elution profile of DEAE Sepharose CL-6B column chro intensifying screen for 6h at -70•Ž. Spots in the mem matography is shown in Fig. 2. We assayed the eluted brane were cut out and the radioactivity was measured with fraction by two methods: dot blot hybridization using a liquid scintillation counter. 5•L [32P]-labeled probe, and acceptor activity of [14C] iso Sequencing of the tRNA„ŸTwo-dimensional (2D) TLC leucine. For the dot blotting, eluted tRNA was directly (Funacel SF plate, Funakoshi) for nucleotide analysis was immobilized on a nylon membrane by UV irradiation and developed with two systems. System A: 1st. isobutyric then hybridized with the probe. The profile reveals that the acid : 0.5M NH4OH=5:3 (v/v), 2nd. 2-propanol: c.HCl: H20=70:15:15 (v/v); system B: 1st. isobutyric acid : 0.5M NH4OH=5:3, 2nd. 0.1M Na2HPO4 (pH 6.8) 100 ml, (NH4)2SO4 60g, propanol 2ml (11, 15). A post-label ing method was primarily used for the confirmation of tRNA species and the search for modified nucleotides. For further confirmation of the modified nucleotides, the purified tRNA (150 A260) was digested with RNase T1 and the digest was applied to urea column chromatography (Sephadex A-25, 0.6cm•~100cm) (16). The oligomer containing the anticodon sequence was digested completely with nuclease PI and analyzed by 2D TLC using system A. Mononucleotides were examined by UV spectroscopy, and the nucleotide mixture located in the same position as adenosine 5•L-monophosphate (pA) was further subjected to a paper electrophoresis [pH 7.5 20mM TEAB (triethylam monium bicarbonate) buffer] . As a result, 0.08 A260 (0.28 A220) of unidentified nucleotide (pN) was obtained. pN was then analyzed by FAB mass spectroscopy. The FAB mass spectrum was recorded utilizing JEOL SX102 spectrom eter with 10kV accelerating voltage, FAB priming energy of 6kV using xenon, and diethanolamine as a matrix. Positive ions were collected and analyzed by DA6000 data system. For the identification of 7-methylguanosine and other modified nucleotides, Sanger's method (2D electro phoresis) was used in a preparative scale together with UV spectroscopy (17, 18). Aminoacylation by B. subtilis S30 Fraction-tRNA (1 ƒÊ g) was aminoacylated in the following buffer (total volume 100ƒÊl): 100mM HEPES (pH 7.2), 10mM Mg(OAc)2, 10 mM KCl, 2mM ATP, [14C]isoleucine (specific activity 387 mCi/mmol, NEN) or [14C]methionine (225mCi/mmol, NEN), 20ƒÊl of S30 fraction (19). After the reaction, radioactivity was measured by the procedure used for E. coli (11). RESULTS In B. subtilis, four isoleucine tRNA genes have been identified so far in rrnA, rrnO (20), and rrnB (6). The two tDNAs with the GAT anticodon, described as tDNA(GAT), have only one different base pair in the T-loop (Fig. 1A). However, the tDNA with the CAT anticodon, tDNA(CAT), differs from the tDNA(GAT) in 16 positions (Fig. 1B). We Fig. 1. Secondary structures of B. subtilis isoleucine tDNAs attempted to distinguish the tRNAs corresponding to the deduced from the genes. A: Major tDNAs and arrows mean different base-pairs between two major tDNAs coded in rrnA, rrnO, and rrnB. two kinds of anticodons by dot blot hybridization. A The major tDNA coded in rrnB has an AT pair in the T-stem. rrnA pentadecadeoxyribonucleotide corresponding to the region and rrnO code identical major tDNA, which has a GC pair in the from 38 to 52 of the tDNA(CAT) was used as a probe T-stem. B: Minor tDNA and outlined letters represent different because 5 of the 16 different nucleotides were concentrated nucleotides from the major one. J. Biochem. Modification of B. subtilis minor tRNA11e 813 peak of the dot blot preceded that of the major accepting tides on 2D TLC (system A) revealed the existence of activity.