Vol. 128, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 671-672 Copyright C) 1976 American Society for Microbiology
Printed in U.S.A.
Escherichia coli Mutant Lacking 4-Thiouridine in Its Transfer Ribonucleic Acid T. V. RAMABHADRAN,' T. FOSSUM, AND J. JAGGER* Biology Programs, University of Texas at Dallas, Richardson, Texas 75080 Received for publication 23 July 1976
A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wildtype cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.
4-Thiouridine (4Srd) is a modified nucleoside present in the transfer ribonucleic acid (tRNA) of many Escherichia coli strains (1, 5). Its presence does not drastically affect the functions of tRNA in vitro (10, 12). There are no reports, however, on the effect of 4Srd in vivo. In this note we report the isolation of a mutant of E. coli that has little if any Srd in its tRNA. We find that it grows at the same rate as wild-type cells. The mutant also shows only slight nearultraviolet (near-UV)-induced growth delay. 4Srd in tRNA absorbs maximally around 340 nm (4). Irradiation at 340 nm of such tRNA in vitro or in vivo leads to the formation of 4SrdCyd adducts (2, 9), which have been shown to affect the ability of some tRNA's to be acylated in vitro. Formation of such 4Srd-Cyd adducts in tRNA, leading to an effective amino acid starvation and cessation of RNA synthesis, appears to be the cause of growth inhibition induced by near-UV radiation (300 to 380 nm) in E. coli (6, 8). Relaxed (rel-) mutants ofE. coli, which continue net RNA synthesis during amino acid starvation, also continue RNA synthesis after near-UV irradiation (unlike rel+ strains) and show only minimal growth inhibition (6, 8). This difference in growth response of rel+ and rel- strains to near-UV irradiation can be used to select for relaxed mutants (7). While standardizing the procedure for the isolation of rel- mutants from E. coli B/r NC32 (lac-, valSts, rel+) using near UV, we found 1 isolate (out of 16) that was resistant to growth inhibition but, contrary to expectation, was also rel+ by the criterion of RNA accumulation during amino acid starvation. This prompted us to examine the 4Srd content of this mutant, named E. coli B/r RJ. tRNA extracted from E. coli B/r RJ showed 1 Present address: Department of Biological Chemistry, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
no detectable absorption around 340 nm. Sodium borohydride reduction of tRNA extracted from RJ cells irradiated with black light (peak emission, 355 nm) failed to reveal the development of a new absorption peak at 385 nm, characteristic of 4Srd-Cyd adducts (3, 9). Irradiated reduced tRNA from RJ cells did not show (Fig. 1) the 385-nm fluorescence excitation maximum or the 440-nm fluorescence emission characteristic of the reduced 4Srd-Cyd adduct (3). Based 380
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WAVELEN'GTH (nml
FIG. 1. Fluorescence excitation (emission at 436 nm) and emission (excitation at 396 nm) spectra (uncorrected) of tRNA extracted from near- UV-irradiated E. coli Bir NC32 --- -) and E. coli Bir RJ (-) and then reduced by NaBH,. Cells were grown in M9 minimal medium and irradiated with a General Electric 15-W BLB black-light lamp. tRNA was e-xtracted and reduced with NaBH, in cacodykate buffer, using procedures of Ramabhadran et al. (9). Both preparations had an optical density of 2.8 at 260 nm, with a 1.0-cm optical path. Fluorescence spectra were recorded on a Hitachi-Perkin-Elmer MPF-2A fluorescence spectrophotometer. 671
672
NOTES
on the fluorescence spectrum, we estimate that, if 4Srd-Cyd adducts are present in the irradiated tRNA of RJ, they represent less than 5% of those in wild-type cells. The loss of absorbance of tRNA at 340 nm and the inability to detect 4Srd-Cyd adducts in near-UV-irradiated tRNA strongly suggest the absence of 4Srd from the tRNA of the RJ strain. That the loss of 4Srd is due to a base substitution in the 8-position (where 4Srd is found) seems very unlikely, as this would require mutations at identical positions in a number of tRNA genes. 4Srd seems to be formed in tRNA by post-transcriptional modification (4), and a more plausible explanation is that the mutation affected the modifying enzyme. The RJ mutant has the same growth rate as the parent NC32 in M9 minimal medium (doubling time, 60 min at 30°C), suggesting that the loss of 4Srd does not confer any disadvantage under these conditions of growth. Furthermore, strain RJ shows even less near-UV-induced growth inhibition than most of the relaxed mutants we have examined. Lastly, the existence of a mutant both lacking 4Srd and being resistant to near-UV-induced growth inhibition provides strong support for earlier proposals (6, 8, 9, 11) that 4Srd is the chromophore for near-UV-induced growth delay. This work was supported by Public Health Service grant AM14893 from the National Institute of Arthritis, Metabolism, and Digestive Diseases.
J. BACTERIOL. LITERATURE CITED 1. Carre, D. S., G. Thomas, and A. Favre. 1974. Conformation and functioning of tRNAs; cross-linked tRNAs as substrate for tRNA nucleotidyl-transferase and aminoacyl synthetases. Biochimie 56:1089-1101. 2. Favre, A., A. M. Michelson, and M. Yaniv. 1971. Photochemistry of 4-thio-uridine in Escherichia coli transfer RNA,Val. J. Mol. Biol. 58:367-379. 3. Favre, A., and M. Yaniv. 1971. Introduction of an intramolecular fluorescent probe in E. coli tRNA. FEBS Lett. 17:236-240. 4. Hall, R. 1971. The modified nucleosides in nucleic acids. Columbia University Press, New York. 5. Lipsett, M. N. 1965. The isolation of 4 thiouridylic acid from the soluble ribonucleic acid of Escherichia coli. J. Biol. Chem. 240:3975-3978. 6. Ramabhadran, T. V. 1975. Effects of near-ultraviolet and violet radiations (313-405 nm) on DNA, RNA, and protein synthesis in E. coli B/r: implications for growth delay. Photochem. Photobiol. 22:117-123. 7. Ramabhadran, T. V. 1976. Method for the isolation of Escherichia coli relaxed mutants utilizing near-ultraviolet radiation. J. Bacteriol. 127:1587-1589. 8. Ramabhadran, T. V., and J. Jagger. 1976. The mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation. Proc. Natl. Acad. Sci. U.S.A. 73:59-63. 9. Ramabhadran, T. V., T. K. Fossum, and J. Jagger. 1976. In vivo induction of 4-thiouridine-cytidine adducts in tRNA of E. coli B/r by near-ultraviolet radiation. Photochem. Photobiol. 23:315-321. 10. Saneyoshi, M., and S. Nishimura. 1971. Selective inactivation of amino acid acceptor and ribosome binding activities of Escherichia coli tRNA by modification
with cyanogen bromide. Biochim. Biophys. Acta 246:123-131. 11. Thomas, G., and A. Favre. 1975. 4-Thiouridine as the target for near-ultraviolet light induced growth delay in Escherichia coli. Biochem. Biophys. Res. Commun. 4:1454-1461. 12. Walker, R. T., and U. L. Rajbhandary. 1972. Studies on polynucleotides. J. Biol. Chem. 247:4879-4892.
JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 671-672 Copyright C) 1976 American Society for Microbiology
Printed in U.S.A.
Escherichia coli Mutant Lacking 4-Thiouridine in Its Transfer Ribonucleic Acid T. V. RAMABHADRAN,' T. FOSSUM, AND J. JAGGER* Biology Programs, University of Texas at Dallas, Richardson, Texas 75080 Received for publication 23 July 1976
A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wildtype cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.
4-Thiouridine (4Srd) is a modified nucleoside present in the transfer ribonucleic acid (tRNA) of many Escherichia coli strains (1, 5). Its presence does not drastically affect the functions of tRNA in vitro (10, 12). There are no reports, however, on the effect of 4Srd in vivo. In this note we report the isolation of a mutant of E. coli that has little if any Srd in its tRNA. We find that it grows at the same rate as wild-type cells. The mutant also shows only slight nearultraviolet (near-UV)-induced growth delay. 4Srd in tRNA absorbs maximally around 340 nm (4). Irradiation at 340 nm of such tRNA in vitro or in vivo leads to the formation of 4SrdCyd adducts (2, 9), which have been shown to affect the ability of some tRNA's to be acylated in vitro. Formation of such 4Srd-Cyd adducts in tRNA, leading to an effective amino acid starvation and cessation of RNA synthesis, appears to be the cause of growth inhibition induced by near-UV radiation (300 to 380 nm) in E. coli (6, 8). Relaxed (rel-) mutants ofE. coli, which continue net RNA synthesis during amino acid starvation, also continue RNA synthesis after near-UV irradiation (unlike rel+ strains) and show only minimal growth inhibition (6, 8). This difference in growth response of rel+ and rel- strains to near-UV irradiation can be used to select for relaxed mutants (7). While standardizing the procedure for the isolation of rel- mutants from E. coli B/r NC32 (lac-, valSts, rel+) using near UV, we found 1 isolate (out of 16) that was resistant to growth inhibition but, contrary to expectation, was also rel+ by the criterion of RNA accumulation during amino acid starvation. This prompted us to examine the 4Srd content of this mutant, named E. coli B/r RJ. tRNA extracted from E. coli B/r RJ showed 1 Present address: Department of Biological Chemistry, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
no detectable absorption around 340 nm. Sodium borohydride reduction of tRNA extracted from RJ cells irradiated with black light (peak emission, 355 nm) failed to reveal the development of a new absorption peak at 385 nm, characteristic of 4Srd-Cyd adducts (3, 9). Irradiated reduced tRNA from RJ cells did not show (Fig. 1) the 385-nm fluorescence excitation maximum or the 440-nm fluorescence emission characteristic of the reduced 4Srd-Cyd adduct (3). Based 380
400
420
440
460
380
400
420
440
460
z z w
320
340
360
480
500
WAVELEN'GTH (nml
FIG. 1. Fluorescence excitation (emission at 436 nm) and emission (excitation at 396 nm) spectra (uncorrected) of tRNA extracted from near- UV-irradiated E. coli Bir NC32 --- -) and E. coli Bir RJ (-) and then reduced by NaBH,. Cells were grown in M9 minimal medium and irradiated with a General Electric 15-W BLB black-light lamp. tRNA was e-xtracted and reduced with NaBH, in cacodykate buffer, using procedures of Ramabhadran et al. (9). Both preparations had an optical density of 2.8 at 260 nm, with a 1.0-cm optical path. Fluorescence spectra were recorded on a Hitachi-Perkin-Elmer MPF-2A fluorescence spectrophotometer. 671
672
NOTES
on the fluorescence spectrum, we estimate that, if 4Srd-Cyd adducts are present in the irradiated tRNA of RJ, they represent less than 5% of those in wild-type cells. The loss of absorbance of tRNA at 340 nm and the inability to detect 4Srd-Cyd adducts in near-UV-irradiated tRNA strongly suggest the absence of 4Srd from the tRNA of the RJ strain. That the loss of 4Srd is due to a base substitution in the 8-position (where 4Srd is found) seems very unlikely, as this would require mutations at identical positions in a number of tRNA genes. 4Srd seems to be formed in tRNA by post-transcriptional modification (4), and a more plausible explanation is that the mutation affected the modifying enzyme. The RJ mutant has the same growth rate as the parent NC32 in M9 minimal medium (doubling time, 60 min at 30°C), suggesting that the loss of 4Srd does not confer any disadvantage under these conditions of growth. Furthermore, strain RJ shows even less near-UV-induced growth inhibition than most of the relaxed mutants we have examined. Lastly, the existence of a mutant both lacking 4Srd and being resistant to near-UV-induced growth inhibition provides strong support for earlier proposals (6, 8, 9, 11) that 4Srd is the chromophore for near-UV-induced growth delay. This work was supported by Public Health Service grant AM14893 from the National Institute of Arthritis, Metabolism, and Digestive Diseases.
J. BACTERIOL. LITERATURE CITED 1. Carre, D. S., G. Thomas, and A. Favre. 1974. Conformation and functioning of tRNAs; cross-linked tRNAs as substrate for tRNA nucleotidyl-transferase and aminoacyl synthetases. Biochimie 56:1089-1101. 2. Favre, A., A. M. Michelson, and M. Yaniv. 1971. Photochemistry of 4-thio-uridine in Escherichia coli transfer RNA,Val. J. Mol. Biol. 58:367-379. 3. Favre, A., and M. Yaniv. 1971. Introduction of an intramolecular fluorescent probe in E. coli tRNA. FEBS Lett. 17:236-240. 4. Hall, R. 1971. The modified nucleosides in nucleic acids. Columbia University Press, New York. 5. Lipsett, M. N. 1965. The isolation of 4 thiouridylic acid from the soluble ribonucleic acid of Escherichia coli. J. Biol. Chem. 240:3975-3978. 6. Ramabhadran, T. V. 1975. Effects of near-ultraviolet and violet radiations (313-405 nm) on DNA, RNA, and protein synthesis in E. coli B/r: implications for growth delay. Photochem. Photobiol. 22:117-123. 7. Ramabhadran, T. V. 1976. Method for the isolation of Escherichia coli relaxed mutants utilizing near-ultraviolet radiation. J. Bacteriol. 127:1587-1589. 8. Ramabhadran, T. V., and J. Jagger. 1976. The mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation. Proc. Natl. Acad. Sci. U.S.A. 73:59-63. 9. Ramabhadran, T. V., T. K. Fossum, and J. Jagger. 1976. In vivo induction of 4-thiouridine-cytidine adducts in tRNA of E. coli B/r by near-ultraviolet radiation. Photochem. Photobiol. 23:315-321. 10. Saneyoshi, M., and S. Nishimura. 1971. Selective inactivation of amino acid acceptor and ribosome binding activities of Escherichia coli tRNA by modification
with cyanogen bromide. Biochim. Biophys. Acta 246:123-131. 11. Thomas, G., and A. Favre. 1975. 4-Thiouridine as the target for near-ultraviolet light induced growth delay in Escherichia coli. Biochem. Biophys. Res. Commun. 4:1454-1461. 12. Walker, R. T., and U. L. Rajbhandary. 1972. Studies on polynucleotides. J. Biol. Chem. 247:4879-4892.
