Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India1
Author for correspondence: Umesh Varshney. Tel: +91 80 394 2686. Fax: +91 80 360 2697. e-mail: varshney{at}mcbl.iisc.ernet.in
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ABSTRACT |
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Keywords: tyrT-tyrV, tyrU, preQ1, preQ0, acid urea gels
Abbreviations: preQ0, 7-cyano-7-deazaguanine; preQ1, 7-aminomethyl-7-deazaguanine
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INTRODUCTION |
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The queuosine in tRNAs has been proposed to modulate the interaction between the anticodon and the degenerate codons (Meier et al., 1985 ), and shown to be important for survival of E. coli in the natural environment (Noguchi et al., 1982
). Interestingly, in Shigella flexneri, vacC, a virulence-associated chromosomal locus, is homologous to tgt of E. coli and a vacC mutant is complemented by tgt from E. coli (Durand et al., 1994
). The mutations in vacC in S. flexneri result in the loss of pathogenicity, suggesting tgt as a possible drug target for shigellosis (Gradler et al., 2001
). On the other hand, many tumours and neoplastic cell lines are deficient in queuosine modification (Aytac & Gunduz, 1994
; Harada & Nishimura, 1972
; Okada et al., 1979
; Randerath et al., 1984
). It has been suggested that queuosine modification in tRNA regulates protein synthesis and influences cellular growth and differentiation in tumour cells (Morris et al., 1999
). The eukaryotes are unable to carry out de novo synthesis of queuine, and as a nutritional factor, queuine has also been suggested to modulate receptor tyrosine kinases (Langgut, 1995
). As the eukaryotes obtain queuine through diet or the gut microflora (Slany & Kersten, 1994
), its analogues could serve as important therapeutic agents to treat cancer or related ailments (Nishimura, 1972
; Nishimura et al., 1983
).
Despite such crucial biological implications of queuosine modification in the tRNAs, the details of the biosynthesis of preQ1 (or preQ0) base are largely unknown. Availability of well-characterized mutants is an important tool in exploring the complex biochemical steps in the biosynthetic pathways. During the course of our studies on structurefunction relationship of initiator tRNA, we discovered that an isolate of E. coli (E. coli B105) lacked queuosine modification. Further characterization of this strain suggests that the lack of queuosine in tRNAs in this strain is due to some defect(s) at the step(s) prior to the transglycosylation step. Using this novel strain, it should now become possible to gain insights into the steps involved in the de novo biosynthesis of the precursors of the base of queuosine.
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METHODS |
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Southern blot analysis.
Genomic DNA (5 µg) from E. coli B105 (a B-strain) and E. coli CA274 (a K-strain) were digested with BstEII or BstNI following the suppliers instructions, resolved on agarose gel (0·9%), transferred onto nytran membrane under vacuum and hybridized with radiolabelled probe against tRNATyr (Reed & Mann, 1985 ).
Cloning of the tyrU gene.
The tyrU locus was PCR amplified from both the E. coli strains, CA274 and B105. Genomic DNA (200 ng) and 40 pmol each of the forward (5'-GGTCACGC- GTTCGATTCCGGTAG-3') and reverse (5'-ACGGATCCATCGGTGATATCACC-3') primers were used in PCR with Vent DNA polymerase (New England Biolabs). The reactions were subjected to initial incubation at 94 °C for 5 min followed by 30 cycles each consisting of 1 min at 94 °C, 1 min at 55 °C and 1·5 min at 68 °C. The PCR products were used for cycle sequencing; the one from B105 was cloned into pTrc99C and referred to as pTrctyrU.
Cloning of queA and tgt genes and generation of an internal deletion in tgt (tgt).
The queA and tgt cistrons from E. coli CA274 and E. coli B105 were PCR amplified using 200 ng genomic DNA, 40 pmol each of forward (5'-AGAATTCATCGATTATATTCTATCC-3') and reverse (5'-CGAATTCAAAGACATCGGGCTA-3') primers and Pfu DNA polymerase (Promega). After initial denaturation at 94 °C for 5 min, DNAs were amplified in 30 cycles each consisting of 1 min at 94 °C, 1 min at 45 °C and 5 min at 68 °C. The 2·5 kb PCR products from B105 and CA274 were eluted from agarose gel, purified, digested with EcoRI and cloned into pTZN (a pTZ19R-derived vector) linearized with EcoRI, and referred to as pTZqueA-tgt(B105) and pTZqueA-tgt(CA274), respectively. Another construct, pTZqueA-tgt1(CA274), was similar to pTZqueA-tgt(CA274), except that it contained the queA-tgt insert in the opposite orientation. pTZqueA-tgt(CA274) was digested with EcoRV to excise an internal segment (200 bp) of tgt and religated to generate pTZqueA-
tgt(CA274).
Modified base analysis.
Exponentially growing cells of E. coli KL16 or B105 harbouring pTrctyrU(B105) were harvested and metabolically labelled with [32P]orthophosphate in low-phosphate medium (Seong & RajBhandary, 1987 ; Thanedar et al., 2001
). The total tRNAs were fractionated on 15% polyacrylamide gels under non-denaturing conditions. The region in the gel corresponding to tRNATyr was localized with the help of ethidium bromide staining of the gel and the marker tRNATyr (a gift from Dr U. L. RajBhandary, Biology Department, Massachusetts Institute of Technology, Cambridge, USA), cut out and eluted in 5x TE containing 5% phenol and 1 M LiCl. The tRNA preparation (enriched for tRNATyr) was further purified by three steps of phenol extraction.
The labelled tRNATyr (25000 c.p.m.) was mixed with 1 µg tRNATyr and digested with 1 unit RNase T2 in 20 mM ammonium acetate buffer (pH 5·0) at 37 °C for 5 h, then subjected to repeated drying under vacuum to remove traces of ammonium acetate. The dried sample was resuspended in 34 µl water and spotted on cellulose sheets for two-dimensional chromatography (Noguchi et al., 1982 ). The thin-layer chromatography was carried out using isobutyric acid/0·5 M ammonia 5:3 (v/v) solvent for the first dimension for 1518 h until the solvent reached the top of the plate. The plates were dried for 12 h and then run in the perpendicular direction in the second solvent system (2-propanol/water/HCl, 14:3:3, by vol.) (Nishimura, 1972
), air-dried and subjected to autoradiography using a BioImage analyser (Fuji Film BAS1800).
Complementation studies.
The constructs (pTZqueA-tgt of CA274 or B105 origin) or the vector control (pTZN) were transformed into tgt mutant (JE7336), tgt+ (JE7334) or queA mutant (JE10651) strains. The tRNA preparations were analysed by Northern blotting.
Growth competition experiments.
The cultures of E. coli CA274 and B105 were started with 0·25% inoculum from overnight cultures in 25 ml LB broth and grown at 37 °C with shaking. Samples were taken periodically to read OD600. In the growth competition assays, overnight cultures of E. coli CA274 and B105 were mixed in equal volumes (zero subculture) for use as inoculum and grown to saturation (first subculture). Inoculum from the first subculture was used to obtain the second subculture; and an inoculum from the second subculture yielded the third subculture. The abundance of CA274 and B105 in the cultures was determined by dilution plating on MacConkeys agar. E. coli CA274 with an amber mutation in the lacZ gene does not ferment lactose in the medium and produces white colonies, whereas E. coli B105, which utilizes lactose, grows as pink colonies.
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RESULTS |
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Expression of the cloned tyrU gene in E. coli CA274 and B105
To gain further insights, we transformed pTrctyrU into E. coli CA274 and B105. The analysis of tRNAs showed that the introduction of tyrU gene on a plasmid resulted in overproduction of tRNATyr in both the strains (Fig. 2c: compare lane 1 with 2 and 3, and lane 4 with 5 and 6). Interestingly, the bulk of the tRNATyr from the CA274 strain still migrated slower than that from the B105 strain (compare lanes 2 and 3 with 5 and 6). Also, overproduction of tRNATyr in CA274 resulted in a faint band just below the major band (lanes 2 and 3), which co-migrated with the tRNATyr of the B105 strain. This is most likely due to lack of modification in a minor population of tRNATyr under these conditions. Taken together, these observations strongly suggest that the mobility differences in tRNATyr are not caused by mutation, and in all probability arise from strain-specific modifications.
Lack of queuosine modification in E. coli B105
Since the initiator tRNAs from the two strains co-migrated on the same gel (Fig. 1a), differential biosynthesis of 2'-O-methylcytidine (Cm), pseudouridine (
), dihydrouridine (D), thiouridine (S) and ribothymidine (rT), which are common to the tRNATyr and the initiators (as well as other tRNAs) was unlikely to be responsible for the faster mobility of tRNATyr from E. coli B105. We therefore focused our attention on queuosine modification, which is present in a set of tRNAs containing the GUN anticodon (encoding Tyr, His, Asp and Asn). The total tRNAs from several E. coli strains were fractionated on acid urea gels and probed for tRNAHis and tRNATyr (Fig. 3a
and b
, respectively). Importantly, in this analysis we included E. coli CA275, which differs from the CA274 strain in that its tyrT locus encodes a tRNA with CUA anticodon (an amber suppressor tRNA, supF).
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Modified base analysis
To confirm that the faster mobility of the His- and Tyr-specific tRNAs in the B105 strain was due to the lack/deficiency of queuosine modification, we carried out a modified base analysis (Noguchi et al., 1982 ) of tRNA preparations enriched for tRNATyr (Fig. 4
). While a spot corresponding to queuosine (Qp) was seen in the control K strain (Fig. 4b
), it was absent from the B105 strain even upon overexposure of the autoradiogram (Fig. 4a
), confirming that the B105 strain lacked queuosine modification. However, other identifiable modified nucleosides were present commonly in both the strains.
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DISCUSSION |
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The biosynthesis of preQ1 or preQ0 in bacteria is thought to result from the action of GTP-cyclohydrolase-like enzyme on GTP (Fig. 6). However, the two enzymes presently known to possess such activity (cyclohydrolase I and cyclohydrolase II) in E. coli are involved in pteridine and riboflavin biosyntheses, respectively, and their role in the biosynthesis of preQ1 or preQ0 has been ruled out. Moreover, a mutant of E. coli (ribA) defective in cyclohydrolase II still had queuosine modification in its tRNA (Slany & Kersten, 1994
). In fact, the only mutations that have been characterized so far in the pathway leading to queuosine modification in tRNA are at the steps of Tgt and QueA (Noguchi et al., 1982
; Reuter et al., 1991
). The E. coli B105 strain thus provides a tool to investigate the very early steps of biosynthesis of the precursor(s) of queuosine base. Importantly, since synthesis of the queuosine base is unique to prokaryotes (Slany & Kersten, 1994
), detailed knowledge of the enzyme(s) involved at this step could make it a novel target to design a new class of antibacterials.
Tgt overproduction is tolerated in E. coli CA274 but not in E. coli B105. The lethal phenotype in B105 may allow one to isolate gene(s) involved in the synthesis of the precursors of the base of queuosine by suppression of the toxic phenotype of Tgt overexpression. The result of the growth competition experiment shown in Fig. 7(b), which was performed to illustrate the reduced fitness of strain B105 due to lack of queuosine modification, would be more valid if carried out with isogenic strains. Nevertheless, our observation that B105 (Q-) fails to compete with CA274 (Q+) suggests that introduction of gene(s) from a library (Q+) into B105, followed by multiple subculturing, may result in enrichment/selection of transformants wherein the resident plasmid confers a growth advantage to the host due to queuosine modification. Thus, characterization of the E. coli B105 strain in this study will be helpful in the elucidation of the queuosine biosynthetic pathway, which may even be a boon to the field of cancer biology (Nishimura et al., 1983
) and contribute to a better understanding of the virulence of Shigella (Gradler et al., 2001
).
Finally, the electrophoretic analysis of tRNAs on acid urea gels has provided a very useful and sensitive tool to separate the various forms (deacylated, aminoacylated or formylaminoacylated) of tRNA in a large number of studies. These gels have also provided crucial insights into the aminoacylation of tRNA with different amino acids (Li et al., 1996 ) or their differential modification status for the 2-methylthio-N6-(
2-isopentenyl)adenosine (ms2i6A) (Mangroo et al., 1995
). In this study, we provide yet another example of discrimination of tRNA species differentially modified for the queuosine base. In fact, detection of mobility differences even for the tRNAs isolated from the queA mutant background suggests that the acid-urea-gel-based separation system can be exploited for wide-ranging applications.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brenner, S. & Beckwith, J. R. (1965). Ochre mutants, a class of suppressible nonsense mutants. J Mol Biol 13, 629-637.
Cayley, S., Record, M. T.Jr & Lewis, B. A. (1989). Accumulation of 3-(N-morpholino)propanesulfonate by osmotically stressed Escherichia coli K-12. J Bacteriol 171, 3597-3602.[Medline]
Durand, J. M., Okada, N., Tobe, T. & 7 other authors (1994). vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of E. coli K-12. J Bacteriol 176, 46274634.[Abstract]
Frey, B., Janel, G., Michelsen, U. & Kersten, H. (1989). Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr. J Bacteriol 171, 1524-1530.[Medline]
Gay, N. J. (1984). Construction and characterization of an Escherichia coli strain with uncI mutation. J Bacteriol 158, 820-825.[Medline]
Gradler, U., Gerber, H. D., Goodenough-Lashua, D. M., Garcia, G. A., Ficner, R., Reuter, K., Stubbs, M. T. & Klebe, G. (2001). A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase. J Mol Biol 306, 455-467.[Medline]
Harada, F. & Nishimura, S. (1972). Possible anticodon sequences of tRNAHis, tRNAAsn, and tRNAAsp from Escherichia coli B. Universal presence of nucleoside Q in the first position of the anticodons of these transfer ribonucleic acids. Biochemistry 11, 301-308.[Medline]
Langgut, W. (1995). Regulation of signaling by receptor tyrosine kinases in HeLa cells involves the q-base. Biochem Biophys Res Commun 207, 306-311.[Medline]
Li, S., Kumar, N. V., Varshney, U. & RajBhandary, U. L. (1996). Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli. J Biol Chem 271, 1022-1028.
Low, K. B. (1968). Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc Natl Acad Sci USA 60, 160-167.[Medline]
Mandal, N. & RajBhandary, U. L. (1992). Escherichia coli B lacks one of the two initiator tRNA species present in E. coli K-12. J Bacteriol 174, 7827-7830.[Abstract]
Mangroo, D., Limbach, P. A., McCloskey, J. A. & RajBhandary, U. L. (1995). An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation. J Bacteriol 177, 2858-2862.[Abstract]
Maxam, A. M. & Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65, 499-560.[Medline]
McCloskey, J. A., Pamela, F. & Crain, P. F. (1998). The RNA modification database 1998. Nucleic Acids Res 26, 196.
Meier, F., Suter, B., Grosjean, H., Keith, G. & Kubli, E. (1985). Queuosine modification of the wobble base in tRNAHis influences in vivo decoding properties. EMBO J 4, 823-827.[Abstract]
Messing, J. (1981). A system for shotgun DNA sequencing. Nucleic Acids Res 9, 309-321.[Abstract]
Morris, R. C., Brown, K. G. & Elliot, M. S. (1999). The effects of queuosine on tRNA structure and function. J Biomol Struct Dyn 16, 757-774.[Medline]
Nishimura, S. (1972). Minor components in transfer RNA: their characterization, location, and function. Prog Nucleic Acids Res Mol Biol 12, 49-85.[Medline]
Nishimura, S., Shindo-Okada, N., Kasai, H., Kuchino, Y., Noguchi, S., Ligo, M. & Hoshi, A. (1983). Characterization and analysis of oncofetal tRNA and its possible application for cancer diagnosis and therapy. Recent Results Cancer Res 84, 401-412.[Medline]
Noguchi, S., Nishimura, Y., Hirota, Y. & Nishimura, S. (1982). Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase. Function and biosynthesis of queuosine in the tRNA. J Biol Chem 257, 6544-6550.
Okada, N., Noguchi, S., Kasai, H., Shindo-Okada, N., Ohgi, T., Goto, T. & Nishimura, S. (1979). Novel mechanism of post-transcriptional modification of tRNA. J Biol Chem 254, 3067-3073.[Abstract]
Randerath, E., Agarwal, H. P. & Randerath, K. (1984). Specific lack of the hypermodified nucleoside, queuosine, in hepatoma mitochondrial aspartate transfer RNA and its possible biological significance. Cancer Res 44, 1167-1171.[Abstract]
Reed, K. C. & Mann, D. A. (1985). Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13, 7207-7221.[Abstract]
Reuter, K., Slany, R., Ullrich, F. & Kersten, H. (1991). Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuosine, a nutrient factor for eukaryotes. J Bacteriol 173, 2256-2264.[Medline]
Romier, C., Reuter, K., Suck, D. & Ficner, R. (1996). Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile. Biochemistry 35, 15734-15739.[Medline]
Rozenski, J., Crain, P. F. & McCloskey, J. A. (1999). The RNA modification database: 1999 update. Nucleic Acids Res 27, 196-197.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Seong, B. L. & RajBhandary, U. L. (1987). Escherichia coli formylmethionine tRNA: mutations in GGG/CCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop. Proc Natl Acad Sci USA 84, 334-338.[Abstract]
Slany, R. K. & Kersten, H. (1994). Genes, enzymes and coenzymes of queuosine biosynthesis in procaryotes. Biochimie 76, 1178-1182.[Medline]
Sprinzl, M., Hartman, T., Weber, J., Blank, J. & Zeidler, R. (1989). Sequences supplement. Nucleic Acids Res 17, r1-r172.
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.[Medline]
Thanedar, S., Kumar, N. V. & Varshney, U. (2000). The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins. J Biol Chem 275, 20361-20367.
Thanedar, S., Dineshkumar, T. K. & Varshney, U. (2001). The mere lack of rT modification in initiator tRNA does not facilitate formylation-independent initiation in Escherichia coli. J Bacteriol 183, 7397-7402.
Timms, A. R. & Bridges, B. (1996). The tyrT locus of E. coli B. J Bacteriol 178, 2469-2470.[Abstract]
Varshney, U., Lee, C. P. & RajBhandary, U. L. (1991). Direct analysis of aminoacylation levels of tRNA in vivo. J Biol Chem 266, 24712-24718.
Received 21 June 2002;
revised 18 August 2002;
accepted 20 August 2002.