WEISS,
KIMES,
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MORRIS
Cations and Ribosome Structure. 111. Effects on the 30s and 50s Subunits of Replacing Bound Mg2; by Inorganic Cationst Richard L. Weiss,$ Brian W. Kimes,S and David R. Morris*
Using equilibrium dialysis, the physical and functional properties of purified ribosomal subunits from Eschericliiu coli were studied as ionically bound Mg2* was replaced by divalent cations, MnZT, Ca?-, Sr2', and BaZT, and the monovalent cations, Kf and NH1+. With the 30s subunit, Mn?- and Ca2+could totally replace Mg2+without impairing phenylalanine polymerizing activity. Sr2+ was only partially efyective in this capacity, and substitution by Ba2+ caused total inactivation as the Mg2+/RNA-Pwas reduced from 0.10 to 0. Loss of function was accompanied by significant increases in sensitivity to pancreatic ribonuclease and decreases i n sedimentation coefficient. All activity could be regained after treating these altered 30s particles in high ionic strength buf'fers at 40". Only MnZTcould totally replace Mg2- bound to the 50s subunit without irreversible alterations in polymerizing activity. Total substitution of Ca2- for Mg*+ resulted in particles with lower activity and small changes in ribonuclease sensitivity and sedimentation coefficient. Sr ?+ and Ba2- totally inactivated the 50s subunit as the Mg2'jRNA-P was reduced from 0.075 to 0.025. With these latter two cations, there was a six- to eightfold increase in ribonuclease ABSTRACT:
A
variety of studies clearly show that multivalent cations are an indispensible component of ribosomes (reviewed in Weiss and Morris, 1973). However, a systematic examination of the specificity of this requirement has not been made. Previous work in this laboratory has shown that at least one class of cation binding sites can be defined on the basis of its specificity (Weiss and Morris, 1970, 1973; Kimes and Morris, 1973). In replacing Mg2- with the polyamines putrescine and spermidine, it was found that approximately 20 of the rRNA phosphate groups must continue to interact with Mg?' for the functional integrity of the particles to be maintained. In light of these results, the present study was undertaken to further circumscribe the limits of the specificity of this cation requirement. Methods Unless otherwise stated, all preparations and procedures were as previously described (Weiss and Morris, 1973; ~
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the Departmetit of Biochemistry, University of Washingtoii, Seattlc, Wnshington 98195. Receiced June 8 , 1972. Supported in part by grants from the Anierican Heart Association (70 646) a n d the Natiniial Institute of General Medical Sciences ( G M 13957) and by a trciining grant from the National Institute of General Medical Sciences ( G M 00052). Taken in part from dissertations presented by R. L. W. a n d B. W. I95% of the wild type level of intracellular putrescine, grows with a generation time which is essentially normal and contains increased levels of Mg2+ (Morris and Jorstad, 1970). This result is also consistent with the concept that the class I1 sites can be filled by either polyamines or M g 2 + . In the case of the class 111 sites, there is clearly enough intracellular K+ present to neutralize these sites (Solomon, 1962; Lubin and Ennis, 1964; Lusk et a/., 1968). Another facet of this high level of K+ is that it would decrease the affinity of the ribosome for Mg2+, thus helping to maintain a sufficient concentration of free Mg2+ for other metabolic processes that require this cation (Wacker, 1969). From studies with a mutant of E . coli, it has been suggested that K+ limitation causes inhibition of protein synthesis which is known to require high concentrations of K- (Ennis and Lubin, 1961 ; Lubin and Ennis, 1964; Conway, 1964; Spyrides, 1964). An alternative interpretation is that during K + limitation, this cation no longer competes with Mg2+ for the class 111 R N A phosphates. Thus, an intolerably low level of unbound Mg2+would be created. BIOCHEMISTRY,
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The fact that polyamines, when substituted for Mg*+ in the class I sites, produce inactivation of the ribosomes, suggests that it is important that a n intracellular balance between these cations be maintained. Thus Mg*+ starvation results in impairment of protein synthesis and eventual breakdown of ribosomal particles (McCarthy, 1962). We would interpret this result to be due to insufficient M g * l for occupation of the class I sites, leading t o inactivation of both the 30s and 50s ribosomal subunits. The resulting ribosome degradation may be a consequence of the increased sensitivity of such particles to attack by ribonuclease described here. Conversely, Ezekiel and Brockman (1968) showed that high concentrations of spermidine in the growth medium inhibit protein synthesis in E. coli. Accordingly, we would suggest that this effect arises from displacement of Mg2' from the critical class I sites by spermidine. One might expect that intracellular control mechanisms might exist to buffer these sorts of efects. In fact, the activity of biosynthetic arginine decarboxylase, the first enzyme in the conversion of arginine to putrescine (Morris and Pardee, 1966), is regulated in CirrtJ by the balance between Mg2+ and polyamines in a fashion which might be predicted from these considerations (Wu and Morris, 1973). The results presented in this series of papers suggest one, rather nonspecific function for polyamines in E. coli. However, the existence of mutants of this organism, which show a specific growth requirement for spermidine, even in the presence of high concentrations of Mg2- in the culture medium, suggests that there are other biological roles for these compounds (Morris and Jorstad, 1973). References Adams, A,, Lindahl, T.? and Fresco, J. (1967). Proc. Nu?. Aclid. Sci. U . S. 57, 1684. Choi, Y . S., andCarr, C . W. (1967),J. Mol. Biol. 23, 331. Cohn, M . , Danchin. A,, and Grunberg-Manago, M . (1969), J . Mol. Biol. 39, 199. Conway, T. W. (1964), Proc. Nut. Acud. Sci. U. S. 51.1216. Edelnian, T. S., Ts'o, P. 0. P., and Vinograd, J . (1960), Biocliiiii. Bioph.rs. Actu 43, 393. Ennis, H. L., and Lubin, M. (19611, Bioc/Iiiii. Biop/r!,s. Acfu 50, 399. Ezekiel, D. H., and Brockman, H . (1968),J. Mol. B i d . 31, 541. Gordon, J., and Lipmann, F. (1967),J. Mol. Biol. 23,23. Hurwitz, C . , and Rosano, C. L. (1967)> J . Biol. Chem. 242. 3719. Ishida, T., and Sueoka, N. (1967), Pwc. Nut. Acud. Sci. U. S. 58,1080, Kimes, B. W. (1971), Ph.D. Dissertation, University of Washington. Kimes. B. W., and Morris, D . R . (1973), Bioclleinistrj, 12. 442.
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