Biochimica et Biophysica Acta, ! 130 (19921333-335 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05JIf)
BBAEXP 90340
333
Short Sequence-Paper
Nucleotide sequences of serine tRNAs from Bacillus subtilis J i t s u h i r o M a t s u g i , H o n g - T i J i a !, K a t s u t o s h i M u r a o a n d H i s a y u k i l s h i k u r a Laboratory of Chemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken (JapanJ (Received l0 February 19921
Key words: Serine IRNA; l-Methyladenosine;5-Melhoxyuridine;Modifiednucleoside;(B. st~btilis) Three B. subitilis serine tRNAs were sequenced including modified nucleosidcs. All the scrinc tRNAs contained l-methyladenosine in the D-loop. As other characteristic modified nuclcosidcs, 5-mcthoxyuridinc was found in the first letter of the anticodon in the tRNA(UGA).
In Bacillus subtilis, three kinds of scrine tRNA (tRNA(UGA), tRNA(GCU) and tRNA(GGA)) have been predicted from tDNA sequences so far [1,2], but their RNA sequences including modified nucleosides have not been determined yet. Here, we report their RNA sequenced including modified nucleotides and sequence comparison between those of B. subtilis and Escherichia colt. Crude tRNA from B. subtilis W168 was prepared as described previously [3] and purified by the combined use of D E A E Sepharose CL-6B, Sepharose 4B and BD cellulose column chromatography. Sequencing of three serine tRNAs was performed by the post-labeling method, and two-dimensional TLC was employed for the identification of each nucleotide [4-6]. in the case of modified nucleosides, the identification was performed by the combined use of ultraviolet and mass spectroscopy [7]. With regard to mass spectroscopy, a FAB ionization method was employed for the determination of molecular weight [8]. As shown in Fig. 1, all three tRNAs had 1-methyladenosine (m~A) at position 22 in the D-loop. in gen-
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleofide Sequence Databases under the following accession numbers, D10008. D10009 and Dl00010 for tRNA(UGA), tRNA(GCU) and IRNAtGGA), respectively. * Presen! address: Department of Biochemistry, SUNY at Stony Brook, Stony Brook, NY, USA. Abbreviations: TLC, thin layer chromatography; s4U, 4-thiouridine" ratA, i-methyladenosine; i6A, N¢'-(A2 isopenteJlyl)adenosine: t~'A, N-[9-(/3-D-ribofuranosylpurine-6-yl)carbamoyl]threonine; mo~U, 5methoxyuridine; T, ribothymidine; ~ , pseudouridine; D, 5,6-dihydrouridine; cmoSU, uridine-5-(,~acetic acid. Correspondence: J. Matsugi, Laboratory of Chemistry, Jichi Medical School, Minamikaw.achi-machi, Tochigi-ken. 329-04, Japan.
eral, l-,ncthyladenosine is seen in the TqtC-loop of eukaryotic tRNAs. In this sense, it is interesting that B. subtilis, which belongs to Gram-positive bacteria, has l-methyladenosine and Gram-negative E. coil does not. Among B. subtilis tRNAs, tyrosine tRNA has been known to have l-methyladenosine and its location in the tRNA is in the same position (position 22) as serine tRNAs in this study [9]. Interestingly, both tRNAs, tyrosine and scrine, belong to class 11 tRNA, which has a long extra-loop, and, from the structure models [10,11], the position around 22 in the D-loop is thought to be close enough to interact with the extraloop region. Thus, l-methyladenosinc might work for stabilization of the interaction of the two loops. As the other modified nucleosidc, 4-thiouridinc (s4U) was found in tRNA(UGA) and tRNA(GGA), in B. subtilis, it has been known that only tyrosine tRNA has this modified nucleoside [9]. From nucleoside composition analysis using two-dimensional TLC, the ratios of 4thiouridine to uridine at position 8 in both tRNAs were 1 : 1 in the case of the tRNA(UGA) and 3:2 in the tRNA(GGA). in the first letter of the anticodon UGA, 5methoxyuridine (mo~U) was found and this modified nucleoside, in B. subtilis, has been identified in tRNA t'r°, tRNAThr, tRNA Ah' and tRNA vat as well [12]. In E. coli, uridine-5-oxyacetic acid (cmo~U) is located at the corresponding position in serine tRNA(UGA) and the above tRNA species (except for thrconinc), it has been reported that cmo~U and mo~U can recognize uridine as well as adenosine and guanosine in the codon-anticodon interaction [13]. Thus, in B. subtilis, the anticodon mo~UGA might bc able to recognize three codons, UCU, UCA and UCG. In comparing these tRNAs with tDNA sequences, two points were inconsistent. One is G-G-G in the
334
a
~H 70
b
C C
nPG.G C G.O A.U G.O GoO
A.U
oUGA.12 A .U U ~CCCAs4U CUCOCUAA II G
'''
G
'''''
,aAGGG GGGGGTO CDGA"~ A 40 ¥ U'Ar, G C
,~
G'C l,G,. C
A, CU'AA"lI~r.'.~ IOA~7 moEJ(, A U
A GU CA
%dlU
C
AOHIO C C
~H lO O O G,O A.U G°O A°U
G.O A•U G.O A.U A.U
G.C 51 W 10GUGA^I2 8 G lOGU GAeI2 80 'G iiA^ "CUOAU 'OCUcooUAA "CCUGsdU UUCCC" "~ G ... '''" A G '" ' .... G ,GGA cAAGGGT~, C C BIAGAG U GoAGGGT¥c Dc e% c 4, DGA ~ G.G A G C'G tt .U ,~5 ~b ab A'U "A .C: G'U G .O C'G G .U C.G 47G.,.G, 'G U ,u G'C ,G oG A q r " C .C C.G C .C 0 A G.G U A G .CU U @A37 bC A I) U A31 G .~, GcU GuA GGA i, ,AA I e
Fig, !. Clover-leaf structures of B, subtilis serine tRNAs. (a) tRNA(UGA): (b) tRNA(CCU); (c) tRNA(GGA). Arrowheads indicate the location of different nucleotide sequence from the tDNA sequence.
extra-loop of tRNA(UGA) (46-47: A in Fig. la), and this region has been reported differently by two groups as G-G-G-G [1] and G-G-G [2]. The other was C in the extra-loop of the tRNA(GCU) (47: D, arrowheads in Fig, lb), and this has been reported as T [9,10]. About this difference, it is possible that these tRNAs were transcribed from an unidentified tRNA gene. Concerning the modified nueleoside 3' adjacent to the anticodon, N~-isopcntenyladenosine (PA) and N[9-(,8-D.ribofuranosylpurin.6.yl)carbamoyl[throonine (t6A) were found in tRNA(UGA) and tRNA(GCU), rcsi~ctivcly, in the case of tRNA(GGA), it is noteworthy that unmodified adenosine is located here, as seen in E, coli serine tRNA(GGA) [14], because tRNAs, which have the codon starting h'om uridine, usually have an isopcntenylated adenosine derivative, in both species, it is though that the anticodon GGA is 'sticky' enough to read UCC and UCU codons and thus, the tRNAs may not require the modification. Fig. 2 shows sequence comparison between the serine tRNAs in B. subtilis and E. coll. it has been reported that the extra-loop has a more important role for scrine accepting activity than the anticodon region [15,16], in B, subtilis, sequence homology in the extraloop region is also observed and, among them, it is interesting that guanosine at 47:C, which is common to all serine tRNA in E. coli [16], is conserved in three R subt///s tRHAs as well. Thus, this guanosine seems to play an important role in charging identity in both species.
A TO O
G-O
~b
:2:...
G,0-O,G% n U
~l'U'l
t~_..IST
Fig. 2. Common sequences to all three B. subtilis tRNAs. Framed nucleotides are common sequence in E. coil and B. subtilis tRNAs.
This work was supported by a Grant-in-Aid from The Ministry of Education, Science and Culture, Japan.
References I Wourousek, E.F., Narasiman, N. and Hansen, J.N. (1984) J. Biol. Chem. 259, 3694-3702. 2 Green, CJ. and Void, B.S. (1983) Nucleic. Acids Res. 11, 57635774. 3 Yamada, Y. and Ishikura, H. (1976) FEBS Lett. 54, 155-158. 4 Kuchino, Y., Kato, M., Sugisaki, H. and Nishimura, S. (1979) Nucleic Acids Res. 6, 3459-3469.
335 5 Kimura, F., Harada, F. and Nishimura, S. (1971) Biochemistry 10, 3277-3283. 6 Silberklang, M., Prochiantz, A., Haenni, A.L. and RajBhandary, U.L. (1977) Eur. J. Biochem. 72, 465-478. 7 Hall, R.H. (1971) The modified Nucleosides in Nucleic Acids, Columbia University Press, New York. 8 McCIoskey, J.A. (1986) in Mass Spectrometry in Biomedical Research (Gaskell, SJ., ed.), pp. 75-95, Wiley, New York. 9 Menichi. B., Arnold, H.H., Heymean, T., Dirheimer, G. and Keith, G. (1980) Biochem. Biophys. Res. Commun. 95, 461-467. 10 Br©nnan, T. and Sundaralingam, M. (1976) Nucleic Acids Res. 3, 3235-3251. 11 Dock-Bregeon, A.C., Westhof, E., Gieg6, R. and Moras, D. (1989) J. Mol. Biol. 206. 707-722.
12 Murao, K., Hasegawa, T. and lshikura, H. (1976) Nucleic Acids Res, 3, 2851-2860. 13 Yokoyama, S., Watanabe, T., Murao, K., Ishikura, It., hamaizumi, Z., Nishimura, S. and Miyazawa, T. (1985) Proc. Natl. Acad. Sci. USA 82, 4905-4909. 14 Grosjean, H., Nicoghosian, K., Haumont, E., $611, D. and Cedergren, R. (1985) Nucleic Acids Res. 13, 5697-5706. 15 Dock-Bregeon, A.C., Carcia, A., Gieg& R. and Moras, D. (1990~ Eur. J. Biochem. 188, 283-290. 16 Himeno, H., Hasegawa, T., Ueda, T., Watanabe, K. and Shimizu, M. (1990) Nucleic Acids Res. 18. 6815-6819.
BBAEXP 90340
333
Short Sequence-Paper
Nucleotide sequences of serine tRNAs from Bacillus subtilis J i t s u h i r o M a t s u g i , H o n g - T i J i a !, K a t s u t o s h i M u r a o a n d H i s a y u k i l s h i k u r a Laboratory of Chemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken (JapanJ (Received l0 February 19921
Key words: Serine IRNA; l-Methyladenosine;5-Melhoxyuridine;Modifiednucleoside;(B. st~btilis) Three B. subitilis serine tRNAs were sequenced including modified nucleosidcs. All the scrinc tRNAs contained l-methyladenosine in the D-loop. As other characteristic modified nuclcosidcs, 5-mcthoxyuridinc was found in the first letter of the anticodon in the tRNA(UGA).
In Bacillus subtilis, three kinds of scrine tRNA (tRNA(UGA), tRNA(GCU) and tRNA(GGA)) have been predicted from tDNA sequences so far [1,2], but their RNA sequences including modified nucleosides have not been determined yet. Here, we report their RNA sequenced including modified nucleotides and sequence comparison between those of B. subtilis and Escherichia colt. Crude tRNA from B. subtilis W168 was prepared as described previously [3] and purified by the combined use of D E A E Sepharose CL-6B, Sepharose 4B and BD cellulose column chromatography. Sequencing of three serine tRNAs was performed by the post-labeling method, and two-dimensional TLC was employed for the identification of each nucleotide [4-6]. in the case of modified nucleosides, the identification was performed by the combined use of ultraviolet and mass spectroscopy [7]. With regard to mass spectroscopy, a FAB ionization method was employed for the determination of molecular weight [8]. As shown in Fig. 1, all three tRNAs had 1-methyladenosine (m~A) at position 22 in the D-loop. in gen-
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleofide Sequence Databases under the following accession numbers, D10008. D10009 and Dl00010 for tRNA(UGA), tRNA(GCU) and IRNAtGGA), respectively. * Presen! address: Department of Biochemistry, SUNY at Stony Brook, Stony Brook, NY, USA. Abbreviations: TLC, thin layer chromatography; s4U, 4-thiouridine" ratA, i-methyladenosine; i6A, N¢'-(A2 isopenteJlyl)adenosine: t~'A, N-[9-(/3-D-ribofuranosylpurine-6-yl)carbamoyl]threonine; mo~U, 5methoxyuridine; T, ribothymidine; ~ , pseudouridine; D, 5,6-dihydrouridine; cmoSU, uridine-5-(,~acetic acid. Correspondence: J. Matsugi, Laboratory of Chemistry, Jichi Medical School, Minamikaw.achi-machi, Tochigi-ken. 329-04, Japan.
eral, l-,ncthyladenosine is seen in the TqtC-loop of eukaryotic tRNAs. In this sense, it is interesting that B. subtilis, which belongs to Gram-positive bacteria, has l-methyladenosine and Gram-negative E. coil does not. Among B. subtilis tRNAs, tyrosine tRNA has been known to have l-methyladenosine and its location in the tRNA is in the same position (position 22) as serine tRNAs in this study [9]. Interestingly, both tRNAs, tyrosine and scrine, belong to class 11 tRNA, which has a long extra-loop, and, from the structure models [10,11], the position around 22 in the D-loop is thought to be close enough to interact with the extraloop region. Thus, l-methyladenosinc might work for stabilization of the interaction of the two loops. As the other modified nucleosidc, 4-thiouridinc (s4U) was found in tRNA(UGA) and tRNA(GGA), in B. subtilis, it has been known that only tyrosine tRNA has this modified nucleoside [9]. From nucleoside composition analysis using two-dimensional TLC, the ratios of 4thiouridine to uridine at position 8 in both tRNAs were 1 : 1 in the case of the tRNA(UGA) and 3:2 in the tRNA(GGA). in the first letter of the anticodon UGA, 5methoxyuridine (mo~U) was found and this modified nucleoside, in B. subtilis, has been identified in tRNA t'r°, tRNAThr, tRNA Ah' and tRNA vat as well [12]. In E. coli, uridine-5-oxyacetic acid (cmo~U) is located at the corresponding position in serine tRNA(UGA) and the above tRNA species (except for thrconinc), it has been reported that cmo~U and mo~U can recognize uridine as well as adenosine and guanosine in the codon-anticodon interaction [13]. Thus, in B. subtilis, the anticodon mo~UGA might bc able to recognize three codons, UCU, UCA and UCG. In comparing these tRNAs with tDNA sequences, two points were inconsistent. One is G-G-G in the
334
a
~H 70
b
C C
nPG.G C G.O A.U G.O GoO
A.U
oUGA.12 A .U U ~CCCAs4U CUCOCUAA II G
'''
G
'''''
,aAGGG GGGGGTO CDGA"~ A 40 ¥ U'Ar, G C
,~
G'C l,G,. C
A, CU'AA"lI~r.'.~ IOA~7 moEJ(, A U
A GU CA
%dlU
C
AOHIO C C
~H lO O O G,O A.U G°O A°U
G.O A•U G.O A.U A.U
G.C 51 W 10GUGA^I2 8 G lOGU GAeI2 80 'G iiA^ "CUOAU 'OCUcooUAA "CCUGsdU UUCCC" "~ G ... '''" A G '" ' .... G ,GGA cAAGGGT~, C C BIAGAG U GoAGGGT¥c Dc e% c 4, DGA ~ G.G A G C'G tt .U ,~5 ~b ab A'U "A .C: G'U G .O C'G G .U C.G 47G.,.G, 'G U ,u G'C ,G oG A q r " C .C C.G C .C 0 A G.G U A G .CU U @A37 bC A I) U A31 G .~, GcU GuA GGA i, ,AA I e
Fig, !. Clover-leaf structures of B, subtilis serine tRNAs. (a) tRNA(UGA): (b) tRNA(CCU); (c) tRNA(GGA). Arrowheads indicate the location of different nucleotide sequence from the tDNA sequence.
extra-loop of tRNA(UGA) (46-47: A in Fig. la), and this region has been reported differently by two groups as G-G-G-G [1] and G-G-G [2]. The other was C in the extra-loop of the tRNA(GCU) (47: D, arrowheads in Fig, lb), and this has been reported as T [9,10]. About this difference, it is possible that these tRNAs were transcribed from an unidentified tRNA gene. Concerning the modified nueleoside 3' adjacent to the anticodon, N~-isopcntenyladenosine (PA) and N[9-(,8-D.ribofuranosylpurin.6.yl)carbamoyl[throonine (t6A) were found in tRNA(UGA) and tRNA(GCU), rcsi~ctivcly, in the case of tRNA(GGA), it is noteworthy that unmodified adenosine is located here, as seen in E, coli serine tRNA(GGA) [14], because tRNAs, which have the codon starting h'om uridine, usually have an isopcntenylated adenosine derivative, in both species, it is though that the anticodon GGA is 'sticky' enough to read UCC and UCU codons and thus, the tRNAs may not require the modification. Fig. 2 shows sequence comparison between the serine tRNAs in B. subtilis and E. coll. it has been reported that the extra-loop has a more important role for scrine accepting activity than the anticodon region [15,16], in B, subtilis, sequence homology in the extraloop region is also observed and, among them, it is interesting that guanosine at 47:C, which is common to all serine tRNA in E. coli [16], is conserved in three R subt///s tRHAs as well. Thus, this guanosine seems to play an important role in charging identity in both species.
A TO O
G-O
~b
:2:...
G,0-O,G% n U
~l'U'l
t~_..IST
Fig. 2. Common sequences to all three B. subtilis tRNAs. Framed nucleotides are common sequence in E. coil and B. subtilis tRNAs.
This work was supported by a Grant-in-Aid from The Ministry of Education, Science and Culture, Japan.
References I Wourousek, E.F., Narasiman, N. and Hansen, J.N. (1984) J. Biol. Chem. 259, 3694-3702. 2 Green, CJ. and Void, B.S. (1983) Nucleic. Acids Res. 11, 57635774. 3 Yamada, Y. and Ishikura, H. (1976) FEBS Lett. 54, 155-158. 4 Kuchino, Y., Kato, M., Sugisaki, H. and Nishimura, S. (1979) Nucleic Acids Res. 6, 3459-3469.
335 5 Kimura, F., Harada, F. and Nishimura, S. (1971) Biochemistry 10, 3277-3283. 6 Silberklang, M., Prochiantz, A., Haenni, A.L. and RajBhandary, U.L. (1977) Eur. J. Biochem. 72, 465-478. 7 Hall, R.H. (1971) The modified Nucleosides in Nucleic Acids, Columbia University Press, New York. 8 McCIoskey, J.A. (1986) in Mass Spectrometry in Biomedical Research (Gaskell, SJ., ed.), pp. 75-95, Wiley, New York. 9 Menichi. B., Arnold, H.H., Heymean, T., Dirheimer, G. and Keith, G. (1980) Biochem. Biophys. Res. Commun. 95, 461-467. 10 Br©nnan, T. and Sundaralingam, M. (1976) Nucleic Acids Res. 3, 3235-3251. 11 Dock-Bregeon, A.C., Westhof, E., Gieg6, R. and Moras, D. (1989) J. Mol. Biol. 206. 707-722.
12 Murao, K., Hasegawa, T. and lshikura, H. (1976) Nucleic Acids Res, 3, 2851-2860. 13 Yokoyama, S., Watanabe, T., Murao, K., Ishikura, It., hamaizumi, Z., Nishimura, S. and Miyazawa, T. (1985) Proc. Natl. Acad. Sci. USA 82, 4905-4909. 14 Grosjean, H., Nicoghosian, K., Haumont, E., $611, D. and Cedergren, R. (1985) Nucleic Acids Res. 13, 5697-5706. 15 Dock-Bregeon, A.C., Carcia, A., Gieg& R. and Moras, D. (1990~ Eur. J. Biochem. 188, 283-290. 16 Himeno, H., Hasegawa, T., Ueda, T., Watanabe, K. and Shimizu, M. (1990) Nucleic Acids Res. 18. 6815-6819.
