Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.     

Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits

Ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where about two sulfhydryl groups per protein were added to the ribosomal particles. The 70S and 30S particles were not treated with elevated concentrations of NH4Cl, in contrast to those used in earlier studies. The modified particles were oxidized to promote disulfide bond formation. Proteins were extracted from the cross-linked particles by using conditions to preclude disulfide interchange. Disulfide-linked protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gels were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Final identification of proteins in cross-linked complexes was made by radioiodination of the proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis. Attention was focused on cross-links between 30S proteins. We report the identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but one of which were found in both 70S ribosomes and free 30S subunits in similar yield. Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21, and S6-S18-S21, have not been reported previously when 2-iminothiolane was used. Cross-links S3-S13, S13-S21, S7-S12, S11-S21, and S6-S18-S21 are reported for the first time. The identification of the seven new cross-links is illustrated and discussed in detail. Ten of the dimers reported in the earlier studies of Sommer & Traut (1976) [Sommer, A., & Traut, R. R. (1976) J. Mol. Biol. 106, 995-1015], using 30S subunits treated with high salt concentrations, were not found in the experiments reported here.     

Localization of proteins S1, S2, S16 and S23 on the surface of small subunits of rat liver ribosomes by immune electron microscopy

Summary Ribosomal proteins S1, S2, S16 and S23 were localized on the surface of the small subunit (40S) of rat liver ribosomes by immune electron microscopy. Antibodies against the single proteins were raised in rabbits and chicken and purified by affinity chromatography. 40S-IgG-40S complexes were obtained by incubation of 40S subunits with non-crossreacting antibodies specific for each of the four proteins and subsequent sucrose density gradient centrifugation. The location of the proteins was determined by means of antibody binding sites visualized in negative contrast in the electron microscope. The four investigated proteins are mainly located in the head region of the small subunit. Exposed antigenic determinants of proteins S1 and S2 were found to be located at different sites of the small subunit whereas proteins S16 and S23 were mapped in a limited region only.S2,S3,S17,S21 according to the new nomenclature (McConkey et al., 1979)     

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

Summary The identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg⁺⁺ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fab's) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgG's specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.Paper No. 84 on Ribosomal Proteins. Preceding paper is by Rahmsdorf et al., Molec. gen. Genet. 127, 259–271 (1973).     

Genetic and biochemical analysis of ribosomal proteins of minocycline-susceptible and -resistant Mycobacterium smegmatis.

A minocycline (MINO)-resistant mutant was isolated from Mycobacterium smegmatis strain Rabinowitschi. Polypeptide synthesis in the cell-free system prepared from the mutant was resistant to minocycline (MINO) because of alterated 30S ribosomal subunits. Upon two-dimensional gel electrophoresis, two proteins of 30S subunit were found to be altered. MINO resistance phenotype was transferred by mating to the recipient strain P-53. MINO resistance phenotype of a recombinant thus obtained was transferred by a different mating system to the recipient strain Jucho, once again. Ribosomal proteins of each of the donors, recipients and recombinants were analyzed and compared on 2-dimensional (2D) electrophoresis. Approximately 50 ribosomal proteins were observed in 70S ribosomes. Some proteins were differently electrophoresed in different strains. The 30S ribosomal subunits contained at least 19 proteins and 50S ribosomal subunits contained at least 23 proteins. Some proteins were easily washed off during dissociation of subunits in sucrose gradients. At least one protein (designated F) in both subunits was observed at the same position. One protein designated C in 30S subunits could be co-transferred to the recipient cells together with resistance phenotype at the frequency of 100% in the 30 recombinants examined so far. The other protein designated D in 30S subunits could be transferred at the frequency of 86-88%. Three other proteins in 50S subunits could be co-transferred to the recipient strain at a lower frequency. Minocycline resistance, therefore, could be mapped close to genes encoding the structure of ribosomal proteins in M. smegmatis.     

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.     

Two-dimensional gel electrophoresis of ribosomal proteins from streptomycin-sensitive and streptomycin-resistant mutants of Chlamydomonas reinhardi.

Ribosomal proteins from three mutant strains of Chlamydomonas reinhardi were analysed and compared by one-dimensional and two-dimensional gel electrophoresis. One mutant was streptomycin-sensitive the other two were streptomycin-resistant, one with a Mendelian the other with a non-Mendelian pattern of inheritance. In the 30-S subunits of chloroplast ribosomes approximately 25 proteins are found and in the 50-S subunits 34 proteins. The 40-S subunits of cytoplasmic ribosomes contain about 31 proteins and the 60-S subunits 44 proteins. The molecular weights of most proteins in all subunits are in the range of 10 000 to 35 000. However, the 60-S subunits contain in addition a protein of molecular weight 50 000 and the 30-S subunits show 6-7 bands of molecular weights from 50 000 to 83 000. The proteins of the cytoplasmic 80-S ribosomes or of their subunits from all three mutants are electrophoretically identical. The proteins of the 70-S organellar ribosomes and both of their subunits show distinct differences between the three strains. Our results indicate that organellar ribosomal proteins are in part controlled by nuclear DNA and in part by organellar DNA.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.     

Co-operative response of oligomeric protein receptors coupled to non-co-operative ligand binding.

The biogenesis of 30 S and 50 S ribosomal subunits in exponentially growing Escherichia coli has been studied by following the rate of appearance of pulse-labelled ribosomal proteins on mature subunits. Cells were pulse-labelled for two minutes and for three and a half minutes with radioactive leucine. Ribosomal proteins were extracted and purified by chromatography on carboxymethyl cellulose and analysed by bidimensional gel electrophoresis. All 30 S proteins and most of the 50 S proteins were thus prepared and their radioactivity counted: unequal labelling was obtained. 30 S and 50 S proteins were ordered according to increasing specific radioactivity at both time pulses. The incorporation was greater at three and a half minutes than at two minutes. No major difference in the order at the two labelling times was observed.Only two classes of proteins can be defined in the 30 S and the 50 S subunits, namely early and late proteins. In each class a gradual increase in the radioactivity is apparent from the poorly labelled to the highly labelled proteins. This suggests a definite order of addition.Early 30 S proteins: S17, S16, S15, S19, S18, S8, S4, S20, S10, S6, S9, S12, S7.Late 30 S proteins: S5, S3, S2, S14, S11, S13, S1, S21.Early 50 S proteins: L22, L20, L21, L4, L13, L16, L3, L23, L18, L24, L28, L17, L19, L29, L32, L5, L15, L2, L30, L27.Late 50 S proteins: L25, L11, L7, L12, L1, L9, L8, L10, L33, L14, L6.This order is discussed taking into account the pool size of the proteins measured in the same conditions of cell culture.     

Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines.

Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50-S ribosomal subunits and reconstituted 30-S particles containing different combinations of 23-S core particles and 30-S subunit split proteins obtained from a polyamine-requiring mutant of E. coli during its growth in the presence or absence of putrescine. It was concluded that the defect in the amount of some kinds of 30-S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine-requiring mutant of E. coli grown in the absence of polyamines. The methylation of 16-S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23-S RNA did not change significantly. The decrease in methylation of 16-S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine. The relationship between the decrease of polypeptide synthetic activity of 30-S ribosomal subunits obtained from a polyamine-requiring mutant of E. coli grown in the absence of polyamines and the decrease of methylation of 16-S RNA is discussed.     

Identification of the amino acid residues of proteins S5 and S8 adjacent to each other in the 30 S ribosomal subunit of Escherichia coli.

When Escherichia coli 30 S ribosomal subunits are reacted with protein-protein bifunctional reagents, a number of protein pairs as well as aggregates containing three or more ribosomal proteins are formed. In the present study we have purified one of the protein pairs obtained by reaction of 30 S ribosomal subunits with either radioactive or nonradioactive dimethylsuberimidate. Following molecular weight determination and ammonolysis, the pair was shown to consist of ribosomal proteins S5 and S8. The "native" structure of the complex was surmised from its capacity to be reconstituted into a biologically active 30 S ribosomal subunit. From peptide maps and primary structure determination of various peptides it was demonstrated that the cross-linking bond between ribosomal proteins S5 and S8 involves primarily the residues Lys-93 of protein S8 and the COOH-terminal lysine (Lys-166) of ribosomal protein S5. This result is substantiated by the finding that a mutant carrying an altered S5 lacking the COOH-terminal lysine yields a greatly reduced amount of S5-S8 cross-link. In addition to the points of cross-linking it was found that Lys-30, Lys-68, and Lys-86 of S8 and Lys-5 of S5 react with dimethylsuberimidate, indicating that these residues are available for reaction and suggesting their topographical localization on the ribosomal surface.     

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined.One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor.Each of the two precursors for the 50 S subunit, P₁50 S and p₂50 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p₁50 S

In contrast to the p₂50 S precursor the p₁50 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl.The precursors p30 S and p₂50 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.     

RNA-protein interactions in the ribosome. II. Binding of ribosomal proteins to isolated fragments of the 16 S RNA

Specific fragments of the 16 S ribosomal RNA of Escherichia coli have been isolated and tested for their ability to interact with proteins of the 30 S ribosomal subunit. The 12 S RNA, a 900-nucleotide fragment derived from the 5′-terminal portion of the 16 S RNA, was shown to form specific complexes with proteins S4, S8, S15, and S20. The stoichiometry of binding at saturation was determined in each case. Interaction between the 12 S RNA and protein fraction $\text{S}\text{16}\text{S}\text{17}$ was detected in the presence of S4, S8, S15 and S20; only these proteins were able to bind to this fragment, even when all 21 proteins of the 30 S subunit were added to the reaction mixture. Protein S4 also interacted specifically with the 9 S RNA, a fragment of 500 nucleotides that corresponds to the 5′-terminal third of the 16 S RNA, and protein S15 bound independently to the 4 S RNA, a fragment containing 140 nucleotides situated toward the middle of the RNA molecule. None of the proteins interacted with the 600-nucleotide 8 S fragment that arose from the 3′-end of the 16 S RNA.When the 16 S RNA was incubated with an unfractionated mixture of 30 S subunit proteins at 0 °C, 10 to 12 of the proteins interacted with the ribosomal RNA to form the reconstitution intermediate (RI) particle. Limited hydrolysis of this particle with T₁ ribonuclease yielded 14 S and 8 S subparticles whose RNA components were indistinguishable from the 12 S and 8 S RNAs isolated from digests of free 16 S RNA. The 14 S subparticle contained proteins S6 and S18 in addition to the RNA-binding proteins S4, S8, S15, S20 and $\text{S}\text{16}\text{S}\text{17}$. The 8 S subparticle contained proteins S7, S9, S13 and S19. These findings serve to localize the sites at which proteins incapable of independent interaction with 16 S RNA are fixed during the early stages of 30 S subunit assembly.     

Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli

The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit. Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups. Mild oxidation led to the formation of disulfide protein-protein cross-links. These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis. Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident. Cross-links between L7/L12 and other ribosomal proteins were also formed. These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17. The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed. Similar experiments were performed with 70 S ribosomes. Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11. The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes. No cross-links between 30 S proteins and L7/L12 were observed.     

Studies on the negative circular dichroic bands around 297 nm of ribosomes from bacterial cells.

The small negative CD bands around 297 nm of isolated 30-S and 50-S ribosomal subunits were precisely measured for three bacteria, Bacillus stearothermophilus, Bacillus subtilis and Escherichia coli Q 13. The intensities of the negative CD bands of 30-S subunits were always much greater than those of 50-S subunits irrespective of the bacterial strains, which may be related to the difference in comformations of rRNAs and proteins in the complexes between these subribosomal particles. The dissociation of 70-S ribosomes into two subunits by lowering Mg2+ concentration caused evident enhancement of intensity of the 297 nm CD band, which was completely reversed by the association of the two subunits into 70-S particles. The melting profiles of CD spectra 3 B. stearothermophilus and E. coli were compared and both subunits of the former were found to be more heat stable than those of the latter. It was found that 5 M urea and 0.5% sodium dodecyl sulfate (SDS) treatment caused considerable reduction of the negative CD intensity around 297 nm of 30-S subunits but no significant change of 50-S subunits, while no significant change was observed for the CD spectra of isolated 16-S and 23-S rRNAs by the same treatment. Effects of EDTA treatment and then addition of Mg2+ on the CD spectra and fluorescence emission spectra of the subunits were also observed and the contribution by the interaction between rRNA s and proteins in ribosomes to the small negative band around 297 nm was discussed.