172 F. LACROUTE AND G. S. STENT

APPEI~LX

Two Regulatory Models Involving Variable Growth Periods of p-Galactosidase

(A) Primary control at transcription level

The i gene product is a repressor which interacts directly with the operator gene. This interaction, which is abolished by the presence of inducer, reduces the rate of rotation of the DNA sector representing the /ae operon, and, since DNA rotation presumably occurs pari pazsu with transcription, the rate of chain growth of the lac messenger RNA chains. Since translation of the operator-pro~mal z cistron of the polycistronic/av messenger RNA can be expected to proceed 10at/Tassu with messenger RNA synthesis, the chain growth-rate of fl-galaetosidase protein would be lower at states of submaximal induction than at mammal induction.

(B) Primary control at translation level

In accordance with the idea first proposed (Yudkln~ 1938), then rejected (Jacob & Monod, 1961), then re-proposed (Gruber & Campagne, 1965; Cline &Bock, 1966), interaction of the nascent fl-galactosidase polypeptide with the "true" inducer molecule, the carbohydrate A, is required before that polypeptide chain can be finished and mature into the intact enzyme molecule. The i gene product is not a "repressor" but an enzyme which transforms the endogenously synthesized A into the non-inducer B and thus prevents spontaneous induction. The action of this enzyme is inhibited by "gratuitous" inducers such as IPTG. Lactose, the "natural" inducer, causes accumulation of a high level of A because it is converted into a product which either inhibits the action of the i gene product or is itseff/~. The growth period of fl-galactosidase protsin is, therefore, inversely related to the intra- cellular concentration of A, the inducer-dependent increment of the minimum growth period representing the average waiting time of the nascent, immature polypeptide chain prior to its interaction with the "true" inducer. Constitutive i - mutants carry a non-functional i gene product and thus accumulate spontaneously high levels of A, where non-inducible i s mutants carry a non-lnhlbitable i gene product and thus do not accumulate high levels of A even in the presence of gratuitous inducers. Operator-constitutive, 0 °, mutants involve a structural alteration of the nascen~ fl-galactosidase polypeptide, which allows its completion without interaction with A.

We are greatly indebted to I. Zabin, J. Brown and J. Fowler for their valuable advice concerning methods of purification and NH2-terrninal amino-acid analysis of small samples of fl-galactosidase.

This investigation was supported by U.S. Public Health Service research grant CA 02129 from the National Cancer Institute.

REFERENCES Anderson, E. H. (1946). Proc. Na~. Acad. S~., Wash. 32, 120. Brown, J. L., Koorajian, S., Katze, J. & Zabin, I. (1966). J. BioL Ghem. 241; 2826. Cline, A. L. &Bock, R. M. (1966). Go/d Spr. Hath. Syrup. Quant. Biol. 31, 321. Craven, G. R., Steers, E. & Anfinsen, C. B. (1965). J. Biol. Ghem. 240, 2468.

~-GALACTOSIDASE P E P T I D E CHAIN G R O W T H 173

Fraenkel-Conrat, H., Harris, J . I . & Levy, A. L. (1955). In Methods of Biochemical A ~ y s i s , ed. by David Glick, vol. 2, p. 359. New York: Interscience.

Gruber, M. & Campagne, R. N. (1965). Kon. ZVederl. Akad. Wetenschappen, Proc. C. 68, 270.

Jacob, F. & Monod, J. (196I). J. Mol. Biol. 3, 318. Kepes, A. & Beguin, S. (1966). Biochim. biophys. Acta, 123, 546. Kolber, A. R. & Stein, W. D. (1966). Nature, 209, 691. Lacrout~, F. & Stent, G. S. (1968). Mo~cuk~r and O6ner~ (Teneti~s, in the press. Maaloe, O. & Kjeldgaard, N. O. (1966). Gontrol of Macromo~-'ulax Synthesis. New York:

W. A. Benjamin. Pardee, A. B., Jacob, R. & Monod, J. (1959). J. Mol. Biol. 1, 165. Sanger, F. (1945). Biochem. J. 39, 507. Schleif, R. (1967). Ph.D. thesis, University of California, Berkeley. Yudkirt, J . (1938). Biol. Rev. Cambridge Phil. •oc. 13, 93.