Escherichia coli ribosomal protein S1 and its mutant, shorter, form ml-S1 were cleaved at internal methionyl residues to yield, respectively, six and five fragments of M , ranging from 1000 to 24000. Methods are described to isolate the fragments in pure form. Four of the fragments (designated F2a, F2b, F3 and F4) contain between 86 and 215 amino acids and are therefore as large as other ribosomal proteins. Fragment F2a, derived from the N-terminal region, has previously been shown to contain the major ribosome binding domain of S1 [S. Giorginis and A. R. Subramanian (1980) J. Mol. Biol. 141, 393-4081, Here we show that the RNA binding domain of S1 is essentially contained in F3 derived from the middle region of S1 and carrying the nonreactive -SH group. The reactive -SH group of S1, whose activity is modified by ligand binding, was localized in F2b, a fragment with little RNA binding capacity.The characteristic RNA binding domain and a weak ribosome binding domain of S1 have previously been localized in the large trypsin-resistent core S1 -FI [T. Suryanarayana and A. R. Subramanian (1 979) J . Mol. Bid. 127, 41 -541. Together these data indicate that two of the key functional domains of S1 are located in two regions of the molecule separated by an open, exposed segment. The present study also revealed that the nonreactive -SH group of S1 becomes reactive in ml-S1 by the loss of the remote C-terminal region in the latter.Protein S1 of the Escherichia coli ribosome is the largest protein component of this organelle, constituting about 8 % of its protein mass [I]. It is a component required for protein synthesis in v i~o and is believed to act primarily at the mRNA binding step [2, 31. Protein S1 is incorporated into the enzyme Qfi replicase when E. colicells are infected with phage QP, but is precise role therein remains to be elucidated [4, 51.Two years ago we found evidence from physical studies [6] and limited proteolytic digestion [7] that protein S1 is organized into at least two domains. A trypsin-resistant core, carrying about 60 of the molecule from the C terminus, contained the characteristic nucleic acid binding domain of S1 [7]. Later, a mutant form of S1 lacking approximately 20% of the chain length from the C terminus but active in protein synthesis and in all other functional tests for S1 in vitro, was isolated [8 -101. The primary ribosome binding domain of S1 was then localized in the trypsin-sensitive, NH,-terminal region of the molecule [Ill.The last study was carried out with a fragment ( M , E 24000) of S1 produced by CNBr cleavage and made functionally active by renaturation [I 11. We have since then isolated for the purpose of functional domain studies, as well as for primary structure determination, all of the CNBr-produced fragments of S1 and its mutant form ml-S1. One of the new fragments, derived from the middle region of S1 and carrying an -SH group, showed evidence of localization of the nucleic acid binding domain therein. The purification and characterization of all fragments ar...
Ribosomal protein S1 contains in its RNA binding domain four repeating, homologous stretches of sequences. Its functionally active mutant form m1-S1 [Subramanian, A.R., & Mizushima, S. (1979) J. Biol. Chem. 254, 4309] contains only three repeating stretches. In order to assess the functional importance of this repeating sequence, we cleaved S1 at its reactive SH group on Cys-349 and isolated a fragment (S1-F4) that has lost two of the homologous stretches but retains all other essential elements. We find that ribosomes reconstituted with S1-F4 instead of S1 are functionally active in translating poly(U) and poly(A) but totally inactive in translating phage MS2 RNA. The significance of this result is discussed vis-à-vis the initiation step in translating natural mRNA, and a functional role for the tetrarepeat of S1 is suggested.
A gentle and efficient method for selectively removing S1 from ribosomes was developed: the S1-free translation system prepared from such ribosomes is stimulated 10-20-fold (depending on the mRNA) by a stoichiometric amount of added purified S1. With this system, we examined the activity of mono- and di-N-ethylmaleimide derivatives of S1 in protein synthesis using synthetic and natural mRNAs and electrophoretic analysis of the translation products. The results show that ribosomes containing such modified S1's are functionally active although at a somewhat lower level (50-80% activity). Since treatment of S1 with N-ethylmaleimide abolishes the helix-destabilizing ability of S1, we conclude that this ability is not primarily responsible for S1's biological function. A new model for the role of S1 is proposed on the basis of the physical, structural, and RNA-binding properties of S1.
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