Search

An analysis of the degree to which the 55 ribosomal protein genes are clustered in polycistronic transcriptional units in E. coli is presented. Three kinetic approaches were applied: a) The labeling kinetics of RNA at different times after addition of rifampicin to a culture of E. coli growing in glucose minimal medium was used to calculate the distribution of transcribing RNA polymerases over different size classes of mRNA operons. The average length of transcriptional units being transcribed at a given time is about 3300 base pairs. Less than 1% of mRNA synthesis originates from transcriptional units longer than 12000 base pairs and only about 10% from units longer than 7500 base pairs. From this the upper limit of the length of ribosomal protein operons can be estimated. b) The rate of ribosomal protein synthesis as a fraction of the rate of total protein synthesis (αr) was measured during the cessation of mRNA synthesis and its decay after rifampicin addition. αr appears to decrease from 0.125 to 0.09 indicating that probably most ribosomal protein transcriptional units are shorter than the average. c) The kinetics of the rates of synthesis of individual ribosomal proteins was analysed after the release of the inhibition by rifampicin in a partially rifampicin resistant strain at 40°C. The rates of synthesis for all of the 40 ribosomal proteins tested reach half their final values at times falling in the interval 1.4–3.5 min, though for the majority it was reached between 1.6–2.6 min.

Amino-terminal sequences of five purified Escherichia coli 30 S ribosomal proteins (S4, S9, S10, S16, and S20) were compared with those of their functionally corresponding Bacillus stearothermophilus ribosomal proteins identified previously by the reconstitution technique. An automatic Edman degradation method was used for sequence determinations. The sequence of the first 30 residues is presented, except that only the first 25 residues are shown for the S20 pair. Substantial (40 to 70%) sequence homologies have been observed in every case. The results show that the pairs of functionally equivalent proteins, previously identified by the reconstitution technique, are also chemically related. Thus, the present chemical studies give further support for the previous conclusion that two ribosomes with different properties, 30 S subunits from E. coli and B. stearothermophilus , have the same fundamental structural organization.

Dimethylsuberimidate was used to crosslink 14C-labeled chain initiation factor 3 to E. coli 30S particles. The crosslinked ribosomal proteins were analyzed by dodecyl sulfate polyacrylamide gel electrophoresis, and one major radioactive aggregate was found corresponding to a molecular weight of 41,000. Ribosomal protein S12 was identified to be crosslinked to IF-3 by immunological cross-reactivity.

Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH4. After incorporation of either 3H or 14C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex.

Initiation factor IF-3 clearly can cause dissociation of ribosomes by binding to 30S subunits and thus shifting the spontaneous equilibrium, but it theoretically may also do so by binding to 70S ribosomes. Previously reported experiments with differentially labeled ribosomes and subunits seemed to eliminate the latter mechanism, but these experiments neglected a substantial decrease in the specific activity of the added subunits resulting from net dissociation of the ribosomes by IF-3. The present experiments eliminate or correct this effect. The results exclude direct action on 70S ribosomes as a significant mechanism of dissociation by IF-3.

Nitrophenylated 5'-adenylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated pA-U-G. After reduction and subsequent bromoacetylation, an A-U-G analog was obtained, which could be used as an affinity label for the ribosomal A-U-G-binding site(s). After incubating the A-U-G affinity label with 70S ribosomes, 30S subunits programmed for initiation-factor-dependent fMet-tRNAMetf binding were obtained. Hence, the A-U-G analog had irreversibly reacted at the ribosomal decoding site. Initiation complexes which were formed with the labeled 30S subunits were puromycin-resistant. Furthermore, GTP hydrolysis, necessary for proper accommodation of initiator tRNA at the ribosomal donorsite, did not function in these complexes. These data indicate that immobilization of A-U-G at the decoding site of the ribosome allows factor-dependent initiator tRNA binding, but impairs accommodation at the donor site. The ribosomal protein(s) to which A-U-G was covalently bound at the decoding site were identified by polyacrylamide gel electrophoresis in the presence of urea or sarkosyl. The predominant affinity-labeled protein was found to be protein S18. Variation of the incubation conditions of the affinity-labeling reaction leads to attachment of A-U-G label to another ribosomal protein, S4, the ram gene product.

Under certain conditions, initiation factor 3 (IF-3) can cause the release of aminoacyl-tRNA bound to 30S ribosomal subunits of E. coli . It is shown that this IF-3-induced aminoacyl-tRNA release cannot be attributed to either nucleolytic attack or competition between IF-3 and aminoacyl-tRNA for the same ribosomal binding site. It was found that the 30S-aminoacyl-tRNA-codon complexes formed in the absence of IF-3 are intrinsically different from those prepared in the presence of IF-3. In the absence of IF-3, the ribosomal binding of aminoacyl-tRNA is a virtually irreversible process, since the bound aminoacyl-tRNA can neither be spontaneously released upon dilution nor exchanged for unbound aminoacyl-tRNA. In the presence of IF-3, the binding of one molecule of IF-3 per 30S ribosome renders the binding of aminoacyl-tRNA reversible upon dilution and promotes exchange between bound and unbound aminoacyl-tRNA. It is suggested that this difference is due to a conformational transition of the 30S ribosomal subunit induced by the binding of IF-3. The possible implications of this finding in relation to the mechanism of action of IF-3 and its functional role in the cell are discussed.

The structure of negatively stained 30 S ribosome-IgG-30 S ribosome complexes (“dimers”) was examined by electron microscopy to locate proteins S5, S13 and S14 on the surface of the 30S ribosomal subunit from Escherichia coli. The attachment points of non-crossreacting antibodies, specific to each of the three ribosomal proteins, were visualized and correlated to distinctive structural features of the 30S subunit. The 30S particle showed a bipartite structure of two globular sections unequal in size and connected by a narrow bridge (“neck”). Protein S5 was located at several sites on the surface of this neck region. Proteins S13 and S14 were localized in the smaller globular section and were found to be in close proximity to one another. A model of the 30 S subunit with the location of the three proteins is presented.

An experimental and theoretical study has been made into the effect of association-dissociation reactions on the sedimentation of the E. coli ribosomal system 50S-30S ⇄ 50S + 30S. It has been found that (a) the sedimentation pattern is strongly dependent on the rotor speed; (b) the ratio of components as measured using high-speed ultracentrifugation (30000–40000 r.p.m.) is independent of rotor speed; and (c) the speed of ultracentrifugation has a strong effect on the sedimentation coefficient of the ribosomal system as determined by the mean square second moment. The results of this paper demonstrate that ribosome sedimentation at low-speed ultracentrifugation is affected by some artefactual processes. A theoretical analysis of the experimental findings has shown that the observed effects cannot be attributed to the effect of the association-dissociation reaction not to the pressure dependence of the equilibrium constant of that reaction. On the other hand, at high-speed ultracentrifugation the ribosomal system sediments as a heterogeneous mixture of non-interacting components. Consequently, the shape of the boundary in this case will reflect the equilibrium composition of the ribosomal system.

The problem of whether the initiation of bacterial protein synthesis involves the obligatory formation of a 30S initiation complex intermediate was examined in a model system with N-acetylphenylalanyl-tRNA as initiator 5RNA nad poly(uridylic acid) as mRNA. The time courses of the formation of the 30S and 70S initiation complex with Escherichia coli ribosomes were measured simultaneously by stopping the reaction with dextran sulfate and differentiating the N-acetylphenylalanyl-tRNA bound to 30S ribosomal subunits from that bound to 70S ribosomes with RNase I, which hydrolyzes N-acetylphenylalanyl-tRNA bound to 30S subunits but not that bound to 70S ribosomes. A maximum in the 30S complex concentration was observed within the first 10-15 sec of the reaction, whereas 70S complex formed formed more slowly with a slight initial time lag. When an analog computer was programmed with rate constants determined separately for the formation of the 30S initiation complex from preformed 30S complex, kinetic curves very similar to the empirical curves were obtained for the entire time course of the reaction. The results show clearly that formation of the 70S complex obeys the kinetic laws for consecutive reactions, and the 30S complex is, therefore, an obligatory intermediate in the initiation of polyphenylalanine synthesis in the model system.

A diagonal polyacrylamide-dodecyl sulfate gel electrophoresis procedure is described. Its utility is related to the use of the reagent methyl-4-mercaptobutyrimidate as a protein-protein crosslinking reagent. Crosslinking with this reagent occurs through the formation of intermolecular disulfide bonds. Oxidized proteins are separated in one dimension by electrophoresis under non-reducing conditions and in the second dimension under reducing conditions. All proteins except those derived from crosslinked species fall on a diagonal. Methods are described for the identification of the separated monomeric components of crosslinked species. The technique has been applied to the 30S ribosomal subunit of Escherichia coli , and the new crosslinked dimer, S4-S13, has been identified.

The location of the ribosomal proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S subunit of E. coli ribosomes was determined by immune electron microscopy. Antibodies (bivalent IgG's) specific for the six ribosomal proteins were used to form 50S subunit dimers (50S-IgG-50S). The dimers were separated from non-bound antibodies and 50S subunit-monomers and larger aggregates by sucrose density gradient centrifugation. The attachment of each of the six immunoglobulins to 50S subunits was visualized directly by electron microscopy using negative staining and correlated with one or more of the structural features of the particle. Each of the proteins was found to occupy a unique position. Proteins L14, L19 and L23 are located in the region of the 50S subunit which interacts with the 30S particle. Proteins L17 and L22 are on the opposite side of the 50S ribosomal subunit, whilst L18 holds a position on one of the lateral protuberances of a crown-like shaped 50S subunit. Two main forms of 50S subunits were seen on the electron micrographs: one of them had a crown-like shape; the second revealed kidney shaped images. It was demonstrated that these two images are only projections of one structure of the subunit that resembled an armchair; each of the forms could be transformed into the other through rotation by 90°. The results allowed the proposal of a three-dimensional model of the 50S subunit with the location of six different ribosomal proteins as illustrated in Fig. 13.

A spin-labeling study, with a nitroxide analog of N-ethylmaleimide, was carried out to investigate the effect of streptomycin on the conformation of ribosomes from E. coli. Spin-labeling of 70S ribosomes and 30S subunits was performed before and after the addition of streptomycin. Streptomycin has no effect if added after labeling, which confirms that the blocking of sulfhydryl groups of ribosomal proteins interferes with the binding of the antibiotic. However, when the antibiotic is added before labeling, there is a decrease in the rotational correlation time of the labels. This result indicates that the binding of streptomycin to ribosomes loosens the ribosomal structure.

The mechanism of action of chain initiation factor 3 in translation was examined by using E. coli 70S ribosomes which were covalently crosslinked with dimethylsuberimidate. Crosslinked ribosomes were inactive in AUG-dependent fMet-tRNA binding, and were not stimulated by IF-3 in poly(U) translation. IF-3 is known to be required for maximal rates of amino acid incorporation with synthetic polynucleotides at 18 mM Mg 2+ . A direct interaction of IF-3 with 70S ribosomes was demonstrated by crosslinking 14 C-labeled IF-3 to 70S ribosomes. The labeled factor was also crosslinked to 30S and 50S ribosomal subunits. A model is presented proposing the mechanism of action of IF-3 on 70S ribosomes.

Neutron scattering measurements done on E. coli 30S ribosomal subunit specimens in which specific pairs of proteins were deuterated have enabled us to estimate the distances between the labeled proteins. The distances between centers of gravity of three protein pairs have been determined: S2-S5 (105 A), S3-S7 (115 A), and S5-S8 (35 A). A method for extracting shape information about these proteins from the neutron scattering profiles is demonstrated. The method shows that S5 and S8 are compact and S2 is extended.

Transcriptional units for ribosomal proteins in Escherichia coli were measured using the ultraviolet sensitivities of the rates of synthesis of individual ribosomal proteins. The ultraviolet sensitivities of gene transcriptions are proportional to the distances from the promoters. The longest transcriptional units for ribosomal proteins are 3.6 x 10(6) of DNA molecular weight corresponding to 1.8 x 10(6) of RNA or to 180 000 of protein. The length would cover 10--12 genes of ribosomal proteins (of an average Mr of 15000-18000).

Ribosomes treated with colicin E3 are impaired, when tested with phage R17RNA as messenger, in amino acid incorporation, binding of fMet-tRNA, and formation of fMet-puromycin. Studies with subunits showed that formation of the 30S initiation complex is inhibited. The results are consistent with the recent findings that the “E3-fragment” (3′ end of 16S RNA) binds to 30S protein S1, and that both are involved in initiation on natural messenger.

Diimidoesters have been used to cross-link neighboring proteins in the 30S ribosomal subunit of Escherichia coli. Two pairs of cross-linked proteins have been identified by several independent techniques; these are S7-S9 and S13-S19. Since it has been shown earlier that these four proteins cooperate during ribosome assembly in vitro (Green and Kurland, 1973), their identification as near neighbors in the ribosome is quite suggestive. Thus, the data indicate that a cluster of proteins which interact with each other during assembly would be, in general, found in close proximity in the functional ribosome. A more detailed discussion of the arrangement of the proteins in the 30S subunit is presented.

Employing the reconstitution system of Traub and Nomura, the temperature dependence of physical and function reconstitution was studied. The data show that the physical formation of 30S particles occurs at temperatures 15° below the maximum temperature for functional reconstitution. This may indicate that the high activation energy required for functional reconstitution is directed toward arrangement of active sites rather than the interaction of proteins with their cognate sites on RNA.

RNA isolated from calvaria of 16- to 18- day-old chick embryos, assayed in rabbit reticulocyte lysates, programs the synthesis of a collagenase-sensitive protein with the molecular weight of collagen pro-α-chains. When RNA labeled with [ 3 H]uridine for 2 hr and chased for 1 or 2 hr was electrophoresed on aqueous polyacrylamide gels, most of the radioactivity not in 28S or 18S rRNA migrated with an apparent molecular weight of about 1,800,000. After oligo(dT)-cellulose chromotography and analysis in 99% formamide gels, this nonribosomal, rapidly labeled calvaria RNA species migrates at 28S-30S and thus has a molecular weight of at least 1,600,000. Both the ability to program the synthesis of collagenase-sensitive protein in reticulocyte lysates and the presence of a single prominent rapidly labeled 30S peak in acrylamide gels strongly support the deduction that there is only one major mRNA species in calvaria and that this species is collagen messenger RNA.

1. 30S and 50S ribosomal subunits of Escherichia coli were treated with N-[2,3-(14)C]-ethylmaleimide and iodo[(14)C]acetamide. 2. The proteins in the native subunits which reacted with the reagents were S1,double dagger S2, S12, S13, S18, S21, L2, L5, L6, L10, L11, L15, L17, L20, L26+28 and L27. 3. Several proteins, such as S1, S12, S14, S18, L2, L6, L10, L11 and either L26 or 28, had thiol groups in an oxidized form and reacted to a greater extent after reduction with beta-mercaptoethanol or dithiothreitol. 4. The total number of thiol groups in 30S and 50S subunits was determined as 16-17 and 26-27 respectively. The total number of thiol groups in each ribosomal protein was also determined. 5. The reaction of 30S and 50S subunits with iodoacetamide under several different conditions established the order of reactivity of thiol groups.

The kinetics of the rate of synthesis of the ribosomal proteins has been determined during a nutritional shift-up in E. coli. Evidence is obtained for a maximal transcription time of 2 min for possible transcriptional units for ribosomal proteins. This is in accordance with the earlier conclusion that ribosomal protein genes are clustered in several relatively small transcriptional units (Molin et al., 1974).

Evidence is presented suggesting that streptomycin binds to 16S RNA or to 30S ribosomal subunits at the same topographical site located on the RNA chain. The equally bactericidal dihydrostreptomycin binds to the same site as streptomycin but with lower affinity. The effect of drug binding to 16S RNA (measured by reconstitution inhibition) is readily reversible, while that of drug binding to 30S subunits (measured by misreading) persists after removal of the drug. Binding of the drug is not a necessary and sufficient reason for killing.

A new type of kasugamycin-resistant mutant has been isolated from E. coli K12, strain AB312 (Hfr, lac,thr,leu,thi,strA,fus ). In a cell-free protein-synthetic system, the resistance is localized in the ribosome but not in the supernatant fraction. On initiation complex formation, the resistance is associated with the washed ribosome but not with initiation factors. In reconstitution of the 30S ribosomal subunit, the resistance is due to the protein(s) but not to 16S RNA. In two-dimensional electrophoresis, protein S2 is deficient in the 30S ribosomal subunit of kasugamycin-resistant mutant. The results indicate that the kasugamycin-resistance is attributed to alteration of ribosomal protein S2.

The MS2 RNA binding capacity of 30S ribosomal subunits, which is lost when protein S1 is removed, can be restored following incubation with interference factor iα and repelleting. Polyacrylamide-agarose gel electrophoresis shows that, under these conditions, a faster moving, non-RNA binding 30S species, which contains no S1, is converted to a slower moving RNA-binding one, having the same mobility as the 30S species that contains protein S1. Factor iα binds to single-stranded RNAs in a pattern that closely resembles the RNA binding pattern of initiation factor IF-3.

The use of EDTA to ‘unfold’ ribosomal sub-particles has been widespread over the last few years (see e.g. [I] for review). The EDTA particles retain most if not all of the ribosomal proteins [2], but it is not yet known whether the proteins in the unfolded particle retain their specific locations on the ribosomal RNA or whether their distribution becomes random. This is obviously an important consideration, when EDTA particles are used for studies on the organization of the ribosomal components. In particular, several authors have reported the isolation of specific ribo-nucleoprotein fragments from EDTA-treated [3-51 or de-salted [6] particles, which would suggest that the proteins remain specifically located on the RNA. However, in our own studies on specific fragments from E. coli ribosomes, we have been unable to obtain fragments from either the 30s or SOS sub-particles in the presence of EDTA which satisfy our rigorous criteria for specificity [7,8]. This has led us to suspect that in EDTA the proteins have lost their specific sites on the RNA, and in this paper we demonstrate that this is indeed the case, by examining the ability of the proteins to exchange between different ribosomal RNA’s or sub-particles.

The “stringent” protein of the ribosome, required for its synthesis of (p)ppGpp, is readily lost during zonal centrifugation. However, enough can be retained to permit its qualitative localization. It is found in native 50S subunits, runoff 70S ribosomes, and polysomes, but not in native 30S subunits. This protein, therefore, appears to be attached to the 50S moiety of the ribosome, and it may be a constant (though easily removed) component of that structure rather than a factor that joins and leaves during the ribosome cycle.

To investigate ribosome topography and possible function, 70S ribosomes of Escherichia coli were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.

Digestion of reconstituted complexes of 16 S RNA and ribosomal protein S4 with pancreatic ribonuclease, followed by resolution of the products on polyacrylamide gels of low ionic strength, gives rise to a protected ribonucleoprotein fragment with a mobility of 7 S. Fingerprinting of the RNA moiety indicates the presence of sequences covering the 5' one-third of the 16 S RNA. After its extraction from the acrylamide gels, the protected RNA from the complex can be separated into two components by sedimentation in sucrose gradients. One of these retains the ability to reassociate specifically with S4, as judged from its stoichiometry of binding in the presence of excess S4 and from its selection of S4 alone from a mixture of unfractionated 30S ribosomal proteins. It spans the same regions of the 16 S RNA as the total protected RNA, but lacks an internal sequence of about 120 nucleotides. This component contains a number of nicks which give rise to discrete bands upon electrophoresis in urea. The principal subfragments include Sections L through F (230 nucleotides), B through I'' (160 nucleotides), and part of section C'' (35 to 40 nucleotides). It is proposed that the binding site for S4 lies within these noncontiguous sequences, in support of the hypothesis that S4 makes multiple contacts with the 16 S RNA.

A complex was isolated containing one 70 S ribosome bound to the RNA of the f2 bacteriophage in the region of the coat gene. Either the f2 RNA and 70 S ribosome or both ribosomal subunits in this complex were differentially labeled with tritiated lysine or phosphorous 32 RNA. Incubation of this complex in a ribosome-free protein-synthesizing system lacking initiating factor and appreciable nuclease activities allowed the synthesis of coat protein and subsequent termination. Using this techique, it was found that termination resulted in the release of ribosome as subunits. Intermediate in this process is a transient 46 S complex composed of the f2 RNA and 30 S ribosomal subunit, presumably the result of the initial release of the 50 S ribosomal subunit. This 46 S complex subsequently breaks down to a free f2 RNA and 30 S ribosomal subunit. The rate of this latter process is faster in the presence of crude initiating factors. These results are discussed in terms of the mechanism of termination and reading of the intercistronic region.

Summary In this paper, we describe in detail the recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli . The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95 percent of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented.

A temperature sensitive mutant of Escherichia coli was found to have two mutational alterations of its ribosomes: one of these was a streptomycin dependent mutation and the other was a suppressor alteration of S4, with a marked structural change. The altered form of S4 was studied in a strain that was constructed so that this alteration was the only one effecting the structure of the ribosome. Here, it was shown that the mutant form of S4 cause a temperature sensitive defect in the assembly of 30S subunits in vivo which is reflected in the inability of this mutant to properly process ribosomal RNA at the restrictive temperatures. An analysis of both transductants and revertants of this mutant show that the suppression of the streptomycin dependence phenotype, temperature sensitivity, and a defect in RNA processing all have their origin in a single mutational event effecting the structural gene for S4.

Radioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium. The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

The proteins S2, S3, S5, and S10 from the 30S ribosomal subunit of Escherichia coli was studied by analytical ultracentrifugation to characterize them in solution and to determine whether isolated protein-protein interactions exist. Such interactions, if specific, may therefore bear some relationship to the spatial organization of the subunit structure. It was found that protein S2 self-associates to a slight extent and that solution mixtures of S2 and S3 contain only enough dimeric species to account for the S2 dimer. Hence, no observable interaction was detected between S2 and S3. Solution mixtures of proteins S5 and S10 revealed a species of molecular weight greater than either protein. The proposal is that S5 and S10 interact with an association equilibrium constant of 7.6 X 10(-5) M-1 at 3 degrees in a Tris buffer at pH 7.4. It was also shown that solution with a 1:1:1 mixture by mass, of S2, S5, and S10 contained a species possessing a molecular weight consistent with a simple ternary complex of the three proteins.

The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA ‡ . The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg2+ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.

The accessibility of single-stranded sequences in 16S RNA in free state and in ribonucleoprotein particles (RNP) to complementary binding with isoplith fractions of oligonucleotides was studied. RNP had different protein composition and corresponded to intermediate stages of E. coli 30S subunit assembly in vitro. Gel-filtration was used to detect the most strong binding. It was found that S4 essentially inhibited the hexamer binding to RNA. 'Core' proteins bound to 16S RNA strongly increased the shielding of single-stranded regions while 'split' proteins insignificantly changed the hexamer binding. Nevertheless evidence is presented that 'split' proteins might also interact directly with 16S RNA in the 30S subunit.

Circular dichroism (CD) and infrared (IR) spectroscopic analyses were performed of 26 and 18S yeast ribosomal RNA's (rRNA) and their specific complex, 30S RNA. The molecular ellipticity coefficients [thota] of 18, 26, and 30S rRNA's were 2.72, 2.63, and 2.65X10(4) degree-cm2/decimole at 264 nm, respectively. The base-pairing contents of 18, 26, and 30S rna's determined by iC), and 70% (32% AU, 38% GC), respectively. These results suggest that 18 and 26S rRNA have very similar secondary structures, and that 30S rna may have a slightly higher base-pairing content than the estimated sum of those of 18 and 26S rRNA's. The biological significance of this phenomenon is discussed in this report.

Five Hundred μg/ml tetracycline was found to inhibit the accumulation of RNA in Escherichia coli cells. RNA synthesis was not totally inhibited, however. On the contrary, by using DNA-RNA hybridization, it was found that 500 μg/ml tetracycline specifically inhibits ribosomal RNA (rRNA) synthesis but allows messenger RNA synthesis to continue. Also, tetracycline specifically inhibits initiation of ribosomal RNA synthesis as determined from rate studies. The inhibition of rRNA synthesis at 500 μg/ml tetracycline was not due to several trivial explanations, that is, stimulation of ppGpp synthesis, inhibition of nucleoside triphosphate synthesis, nor was it due to sequestering of magnesium ion necessary for RNA polymerase action. Also, 500 μg/ml tetracycline was not inhibitory to RNA synthesis in permeabilized cells capable of normal rates of rRNA synthesis, similar to data previously obtained using ppGpp. Thus, it is proposed that tetracycline stops rRNA synthesis by inhibiting a ribosomal specific transcription factor; this factor is lost or destroyed during cell permeabilization. It is further proposed that tetracycline acts in a manner analogous to the normal rRNA regulator molecule (very likely ppGpp) at the transcriptional level.

Today the number of proteins [l-5] in eukaryotic ribosomal subunits as well as their molecular weights [6] are fairly well determined. Many of these proteins are also isolated and purified [3]. Just like histones, ribosomal proteins are very basic. Both kinds occur naturally in combination with nucleic acids: so it is likely that electrical charges play an important part in the association of these proteins to their respective nucleic acids. As a step towards the understanding of this association it seems therefore necessary to evaluate the electrical charges on the proteins. This may be done globally by measuring their isoelectric points. As the isolation of individual ribosomal proteins is an elaborate and time consuming process, the two-dimensional electrophoresis method of Kaltschmidt [7], which does not require isolation of individual proteins, is used to this end.

The nomenclature proposed by Otaka et al. (1968) for the 30S ribosomal protein components of Escherichia coli as separated by carboxymethyl(CM)-cellulose column chromatography was adopted in several papers in which the genetic loci for many 30S ribosomal proteins on the E. coli chromosome were determined. In order to compare these data with those obtained in other laboratories, the 30S ribosomal proteins fractionated by CM-cellulose chromatography were correlated with the standard nomenclature proposed by Wittmann et al. (1971).

Polyuridylic acid becomes covalently linked to the 30 S subunit of the ribosome when the polymer—ribosome complex is irradiated with ultraviolet light. The polymer bonds to 16 S RNA and also appears to attach to ribosomal proteins.