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Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purified preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10 mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA). [ABSTRACT FROM AUTHOR]

A resistance gene, carB, originally isolated from the carbomycin-producing organism. Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug. However, ribosomes from S. lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides. [ABSTRACT FROM AUTHOR]

Studies the efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes induced by low-intensity and high-intensity photons. Observations of biphotonic processes in the high-intensity photon environment; Efficiency of cross-linking is 20-100 higher upon two-step excitation compared with single-step excitation.

The procedure of isolation and renaturation of all ribosomal proteins from the 30-S subunit of Escherichia coli ribosomes is described. Absorption spectra of these proteins in the near-ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content. Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones. The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins. Circular dichroic spectra of 21 individual proteins from the 30-S subunit of E. coli ribosomes were measured in the range 184–310 nm. The secondary structure of the proteins studied was calculated from the spectra in the range 190–240 nm. Almost all proteins (except proteins S12. S17, S18 and S19) are characterized by a high content of secondary structure. Circular dichroic spectra in the near-ultraviolet region (240–310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied. Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far-ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins; these characteristics may be useful for comparison of protein preparations obtained by different methods in laboratories. [ABSTRACT FROM AUTHOR]

Ribosomal protein methylation has been well documented but its function remains unclear. We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3. This mutant exhibits a cold-sensitive phenotype: its growth rate at 22 °C is abnormally low in complete medium. in addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50-S and 30-S subunits are formed, but at a lower rate. Once assembled, ribosomes with unmethylated L3 are fully active by several criteria. (a) Protein synthesis in vitro with purified 70-S prmB2 ribosomes is as active as wild-type using either a natural (R17) or an artificial [poly(U)] messenger. (b) The induction of β-galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation. (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl. Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA. We suggest that the L3-specific methyltransferase may qualify as one of the postulated 'assembly factors' of the E. coli ribosome. [ABSTRACT FROM AUTHOR]

The function of protein L1 in protein biosynthesis has been examined using ribosomes from two independently derived mutants of Escherichia coli. both of which lacked this protein. In systems in vitro with phage MS2 RNA or poly(U) as message, the mutant ribosomes showed from 40% to 60% of the activity of wild-type ribosomes. The reduction in activity was apparent in the kinetics of [14C]phenylalanine incorporation throughout the incubation period. The activities were restored fully to the wild-type level by the addition of purified protein L1. These results show on the one hand that protein L1 is not essential for protein biosynthesis but, on the other, its presence can significantly increase the overall rate of this process. The data further indicate it as likely that protein L1 exerts its effect at the elongation step. [ABSTRACT FROM AUTHOR]

Protein L11 was isolated from the 50-S subunit of Escherichia coli ribosomes, using two salt extractions and two chromatographic separations on CM-cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands. The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Staphylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro-dansyl-Edman degradations and automatic methods. The N-terminal residue of the protein is blocked and was not degradable in the liquid-phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry. Protein L11 contains three methylated amino acid residues, a Nα-trimethylalanine, and two residues of Nϵ-trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins. [ABSTRACT FROM AUTHOR]

The effects of ultraviolet irradiation on Escherichia coli 30-S ribosomal subunits were studied. At the doses of radiation used in this work (0–4.5 ⊗ 105 quanta/30-S subunit), only protein S7 was found to be significantly crosslinked to the 16-S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30-S subunits to reassociate with 50-S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irrradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16-S RNA by irradiating 30-S subunits depleted in protein S1 or 70-S ribosomes. The covalent complex of 16-S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A-C-C-U-C-G [position 1261–1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399–410]. Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence. [ABSTRACT FROM AUTHOR]

Ribosomes from vegetative cells (vegetative ribosomes) of Bacillus subtilis synthesize guanosine 3′(2′)-diphosphate 5′-diphosphate (p2Gp2) and guanosine 3′(2′)-diphosphate 5'′triphosphate (p3Gp2). In contrast, ribosomes isolated from sporulating cells (sporulation ribosomes) synthesize adenosine 3′(2′)-diphosphate 5′-diphosphate (p2Ap2) and adenosine 3′(2′)-diphosphate 5′-triphosphate (p3Ap2). The products obtained with the aid of vegetative ribosomes were characterized by comparison with authentic substances in two chromatographic systems. The reaction mechanism is identical with that found in Escherichia coli, since [³H]GTP or GDP is pyrophosphorylated by ATP to form p2Gp2 and p3Gp2. Omission of ATP completely stops the formation of any reaction products. Furthermore, no reaction products are observed when GTP or GDP are omitted in a reaction mixture containing vegetative ribosomes. In contrast, incubation of sporulation ribosomes with ATP alone or ADP and ATP results in the formation of two products identified as p2Ap2 and p3Ap2 by their mechanism of synthesis as well as chemical and enzymatic degradation. Sporulation ribosomes (isolated at least an hour after the end of logarithmic growth) synthesize drastically less (by a factor of 100) p2Gp2 and p3Gp2. Vegetative ribosomes, on the other hand, synthesize only 1/100 the amount of p2Ap2 and p3Ap2 compared to sporulation ribosomes when isolated at least an hour before the end of logarithmic growth. By using washed ribosomes and ribosomal wash from vegetative and sporulation ribosomes, we were able to show that changes in the composition of ribosomes (probably in ribosomal proteins and/or ribosome-associated factors) must occur during the transition from vegetative to sporulating cells. Washed ribosomes from vegetative cells cannot replace washed ribosomes from sporulating cells to synthesize p2Ap2 and p3Ap2 in the presence of ribosomal wash from sporulating cells and vice versa. During the transition from one type of ribosomes to the other, as far as the unusual nucleotide-synthesizing activity is concerned, both types of nucleotides can be synthesized. While the ability to synthesize p2Gp2 and p3Gp2 decreases approximately at the end of logarithmic growth, the ability to synthesize p2Ap2 and p3Ap2 increases at the same time. The reason behind this obvious change in ribosomal structure and associated factors is discussed in the light of our finding that these changes are not observed in some early blocked asporogenous mutants. The observation that ribosomes from sporulating cells synthesize traces of p2Gp2 and p3Gp2 (by a factor of almost 100 less than vegetative ribosomes) is probably due to the presence of some non-sporulating cells in any B. subtilis culture. Likewise, there are always some spores contained in a vegetatively growing culture, which are probably responsible for the observed small amounts of p2Ap32 and pa3Ap2 synthesized. [ABSTRACT FROM AUTHOR]

Possible alteration in the ratio of the long and short forms of initiation factor IF-3 (FEBS Lett. 79, 264-275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319-325, 1972), the total amount of IF-3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithrnic phase cells. The activity of IF-3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF-3/ribosome is maintained constant. The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth-cycle-dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se. [ABSTRACT FROM AUTHOR]

Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 and S19, together with RNA from sections O′-D-E′-K-P-P′-E-A of the 16-S sequence. I. When 30-S subunits were hydrolysed with ribonuclease T1 in the presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S 19~ together with minor amounts of S 13 or S 14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections O′-E′ and P-A of the 16-S RNA~ but that section K (from the middle of this area) was missing. 2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S 7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits, the unbound proteins were removed, and RNA fragments containing covalently linked protein S 7 were identified. It was possible to demonstrate that the site of cross-linking lies within the O′-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region. 3, When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences~ but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections O′-E′ and P-A, but that section K was missing (cf. first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein. [ABSTRACT FROM AUTHOR]

50-S subunits were washed with LiCl solutions of different concentrations. After washing with 1 M LiCl solution the particles lost their ability to attach either to 30-S subunits or to the AUG · 30-S subunit · fMet-tRNAfMet complex or to a poly(U) · 30-S subunit · Phe-tRNAPhe complex. Those proteins which were removed by LiCl were fractionated on a Sephadex G-100 column. Of the fractionated proteins only the combinations L1 and L11 or L1 and L16 were essential for the association of 50-S 1.0 cores (particles prepared by washing 50-S subunits in 1.0 M LiCl) with 30-S subunits. These three proteins were also required for the formation of a stable complex between 50-S 1.0 cores, mRNA, 30-S subunits and aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]

Titration of isolated Escherichia coli ribosomal 30-S particles with [14C]phenylalanyl-tRNA in the presence of poly(uridylic acid) was used for a quantitative assay of codon-dependent binding of aminoacyl-tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of "active" 30-S subparticles capable of forming the 30-S · poly(U) · phenylalanyl- tRNA complexes and the equilibrium constants of phenylalanyl-tRNA binding in different media. Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed. The stability of the 30-S · poly(U) · phenylalanyl-tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K+ concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature. It is demonstrated that the fraction of actively binding 30-S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K+) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol. [ABSTRACT FROM AUTHOR]

A method is presented to isolate in pure form two acidic proteins, indicated as A1- and A2-protein from 50-S ribosomes of Escherichia coli. The advantage of this method is that it does not require prior isolation of subunits. The molecular weight of A1- and A2-protein was found by dodecylsulfate gel electrophoresis to be 11 500 with a maxum uncertainty of 1500. Sedimentation equilibrium centrifugation in 5 M guanidine hydrochloride indicates a molecular weight in the range of 12 000-13 000. The amino acid composition of the two proteins is remarkable and indistinguishable. A-protein has a high content of alanine and does not contain tyrosine, tryptophan, histidine or cysteine. On gel-electrofocusing the isoelectric points of A1- and A2-protein are slightly different, namely pH 4.7 and pH 4.85. The amount of A-protein in actively diving cells indicates that contrary to 30-S ribosomes, 50-S ribosomes have a restricted element of repeat. The number of copies of A-protein is found to be somewhat larger than two. [ABSTRACT FROM AUTHOR]

Free N-carbobenzyloxy-phenylalanyl-tRNA is rapidly cleaved by the enzyme peptidyl-tRNA hydrolase. When bound to a 30 S ribosome in the presence of poly(U), the substrate is hydrolyzed as rapidly as when free. The addition of 50 S ribosomal subunits to form the 70 S ribosomal binding complex protects the bound substrate from the enzyme. The degree of protection depends on the state of activity of the 50 S subunit. Under conditions favoring the formation of 70 S ribosomes, active 50 S subunits (capable of catalyzing the peptidyl transferase reaction) afford complete protection at one mole per mole of 30 S subunits Inactive 50 S sub- units also protect the substrate, but less effectively. The protection of bound substrate can be used to measure the amount of 70 S ribosomal binding complex formed and the rate of its formation. Using highly active hydrolase preparations, we have developed a rapid assay procedure and present several examples of its use Under conditions favoring the association of ribosomal subunits, the formation of the 70 S complex may reach completion within a few seconds. [ABSTRACT FROM AUTHOR]