The rimI gene of Escherichia coli K12, which encodes an enzyme catalysing acetylation of the N-terminal alanine of ribosomal protein S18, has been cloned into a mini-F plasmid pRE432 and characterized at the molecular level. Similarly, the rimJ gene, which encodes another acetylating enzyme that is specific for ribosomal protein S5, has been cloned and characterized. From the nucleotide sequence data for the two genes the RimI enzyme was deduced to contain 161 amino acid residues with a calculated molecular weight (Mr) of 18232 and the RimJ enzyme contains 194 amino acid residues with a calculated Mr of 22687. The proteins produced from the two genes in maxi-cells were identified by electrophoresis on acrylamide gels and their operon structure was analysed by insertional mutagenesis with transposon gamma delta (Tn1000) and by measuring the size of their transcripts. Their structural homology was analysed by DNA hybridization and by calculation with computer programs. There is only a low level of overall homology between the two genes except for a 3' terminal region in which a significant degree of homology was noticed.

Ribosomal protein S6 of wild-type strains of Escherichia coli contains up to six glutamic acid residues at its C-terminus. The first two residues are encoded by the structural gene for this protein (rpsF) and the rest are added post-translationally. Mutants deficient in this modification were isolated and characterized genetically and biochemically. The S6 protein in these mutants appeared to contain only two glutamic acid residues at the C-terminus as expected. The mutated gene was termed rimK and was mapped at 18.7 min between cmlA and aroA. The rimK gene was cloned into a cosmid vector and its nucleotide sequence determined. Analysis of the transcriptional and translational products of this gene indicates that it encodes a protein with an Mr of 31.5 kDa and that it forms an operon with a gene encoding a 24 kDa protein. An rpsF mutant containing a Glu to Lys replacement in the second residue from the C-terminus of protein S6 was isolated. The S6 protein of this mutant was apparently inaccessible to the RimK modification system. This indicates that the RimK modification system requires the wild-type amino acid sequence at least in the C-terminal region of ribosomal protein S6.

Temperature-sensitive mutants of an Escherichia coli K-12 strain PA3092 have been isolated following mutagenesis with nitrosoguanidine, and their ribosomal proteins analyzed by two-dimensional gel electrophoresis. This method was found to be very efficient in obtaining mutants with various structural alterations in ribosomal proteins. Thus a total of some 160 mutants with alterations in 41 different ribosomal proteins have so far been isolated. By characterizing these mutants, we could isolate not only those mutants with alterations in the structural genes for various ribosomal proteins, but also those with impairments in the modification of proteins S5, S18 and L12. Furthermore, a mutant has been obtained which apparently lacks the protein S20 (L26) with a concomitant reduction to a great extent of the polypeptide synthetic activity of the small subunit. The usefulness of these mutants in establishing the genetic architecture of the genes coding for the ribosomal proteins and their modifiers is discussed.

The 30S ribosomal subunit of Bacillus stearothermophilus migrated as a single band when electrophoresed on agarose-acrylamide composite gels. The addition of the ribosomal protein SI purified from Escherichia coli resulted in the appearance of an additional band migrating more slowly; "Clabeled SI of E. coli was shown to be associated only with this form. Antibody against E. coli protein SI did not crossreact with either the total 30S ribosomal proteins or the postribosomal supernatant from B. stearothermophilus. These results indicate that B. stearothermophilus lacks a protein equivalent to E. coli SI and may explain our previous finding [Eur. J. Biochem. 56, 15-22 (1975)] that E.coli SI greatly stimulated the translation by B. stearothermophilus ribosomes of f2 phage RNA.In our previous paper (1) we showed that the addition of the 30S ribosomal protein SI purified from Escherichia coli caused a marked stimulation in the in vitro synthesis by Bacillus stearothermophilus ribosomes of coat protein and replicase in response to f2 phage RNA. Without this protein, B. stearothermophilus ribosomes synthesized only A-protein (maturation protein) and a trace amount of coat protein and replicase. Protein Si is the largest of all the ribosomal proteins of E. coli (2) and has been considered to be a fractional protein, that is, a protein present less than one copy per ribosome (3, 4). Recently, this protein was shown to be indispensable for the translation of natural as well as synthetic messengers (5, 6) and for the binding of MS2 phage RNA to ribosomes (7). The 30S ribosomal subunit of E. coli was shown (7-9) to exist in at least two forms, a fast migrating form (F-3OS) and a slow migrating form (S-30S), which were separable from each other by electrophoresis on agaroseacrylamide composite gels. The protein Si converted F-30S into S-30S by binding to the S'-end of 16S RNA (9). The difference between S-30S and F-SOS was thus concluded to be only the presence in the former and absence from the latter of the protein Si. MS2 phage RNA was found (7) to bind only to the former..In this paper, we report that B. stearothermophilus SOS ribosomes exist only in F-30S form and E. coli Si converts them into S-30S form. When incubated in vitro, 4CG-labeled E. coli SI was found to be associated with S-30S form and f2 phage RNA was found to bind preferentially to this form. It thus seems that the 30S ribosomal subunit of B. stearothermophilus does not have a protein functionally equivalent to E. coli Si.MATERIALS AND METHODS Preparation of Ribosomes, f2 Phage and Ribosomal SI. Ribosomes were prepared from B. stearothermophilus strain 799 and from E. coli Ai9 and RNA was extracted from f2 phage particles as described (1). The 30S ribosomal protein Si and Si-depleted 30S subunit from E. coli Ai9 were prepared as described (5, 10). The purity of Si preparation was examined by dodecylsulfate-gel electrophoresis and found more than 95% pure. Purified protein Si and B. stearothermophilus SOS ribosomes were labeled as reported (11, 12...

Twenty-three proteins were identified by two-dimensional electrophoresis on polyacrylamidegel slabs in the 30-S ribosomal subunit of Bacillus stearothermophilus strain 799. They were designated as B-S1 through B-S21, B-Sa and B-Sb and purified on carboxymethyl-cellulose and Sephadex GlOO in the presence of 6 M urea. Their molecular weight was estimated by dodecylsulfate-gel electrophoresis and their amino acid composition was determined after acid hydrolysis. Results obtained for the individual proteins were essentially similar to those for Escherichia coli 30-S proteins with some characteristic differences. [8] showed the restoration of biologically active 30-S ribosomes by addition of the individual proteins purified from B. stearothermophilus to the reconstitution system consisting of E. coli RNA and proteins lacking one particular protein in question. Thus they showed that the E. coli proteins, except for S1, S6, S9, and S13, can be substituted by one of the B. stearothermophilus proteins.As an initial step to elucidate to what extent the ribosomal proteins from a taxonomically distant bacterium, B. stearothermophilus, differ from those of E. coli and to which protein(s) the known functional difference [2 -41 can be assigned, we undertook the purification of all the 30-S ribosomal proteins from B. stoarothermophilus strain 799 to near homogeneity and determined their mobility in two-dimensional gel electrophoresis, molecular weight and amino acid Abbreviations. In this paper B. stearothermophilus and E. coli proteins are designated as B-Sl, B-S2 etc., and E-Sl, E-S2 etc. respectively. composition. In our previous preliminary report [9]we showed the existence of a high degree of similarity between the 30-S ribosomal proteins from B. stearothermophilus and E. coli. Here we report the details of these experiments and some new results. MATERIALS AND METHODS Purification of Ribosomes and Ribosomal ProteinsThe procedure of Hindennach et al.[lo] for purifying the E. coli ribosomal proteins was mainly followed. Washed cells were disrupted by grinding with alumina powder (Alcoa), extracted with buffer I (10 mM potassium phosphate, pH 7.5; 1 mM MgC12; 6 mM 2-mercaptoethanol) and centrifuged at 16 000 rev./min for 30 min. The resultant low-speed supernatant was dialyzed against two to three changes of buffer I for 3 to 4 h and then applied to a 6 % to 38 % (w/w) sucrose gradient in buffer I in a Spinco zonal rotor model B XV. Subunits were separated by centrifugation at 25000 rev./min for 18 h and recovered from the pooled fractions by centrifugation after adjusting the MgC12 concentration to 10 mM. Ribosomes were redissolved in buffer I1 (10 mM Tris-HC1, pH 7.8, 10 mM MgC12, 30 mM NH,CI, 6 mM 2-mercaptoethanol), clarified by centrifugation and stored at -70 "C until use. Proteins were subsequently extracted from the 304 ribosomes thus obtained and separated first on CM-cellulose as described [lo].Eur. J . Biochem. 50 (1975)