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In this paper the question was studied whether Escherichia coli ribosomes which have undergone a specific cleavage of their 16-S rRNA by treatment with the bacteriocin cloacin DF13, are able to enter and complete an elongation cycle. As we have shown in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes with MS2 RNA as a messenger. Binding of the second aminoacyl-tRNA (alanyl-tRNA) to the latter complexes led to the formation of fMet-Ala-tRNA which was fully puromyein-sensitive. In addition to these aminoacyl-tRNAs also the third aminoacyl-tRNA (seryl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycin released all radioactivity from the ribosomes. This indicates that fMet-Ala-Ser-tRNA is formed and is translocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in a 1:1:1 ratio. The defective particles bind fMet-tRNA, Ala-tRNA and Ser-tRNA in a ratio of 1:0.5:0.25. Kinetic experiments strongly suggest that at each binding of the ternary complex aminoacyt-tRNA · EF-Tu · GTP to the A site of defective ribosomes, about half of these particles undergo irreversible inactivation. Consequently polypeptide synthesis under our conditions will come to an end after incorporation of only four or five amino acids. The particles which temporarily survive ternary complex binding are fully capable of transpeptidation and translocation. [ABSTRACT FROM AUTHOR]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit. [ABSTRACT FROM AUTHOR]

The puromycin analogue. 5'-O-(N-bromoacetyl-p-aminophenylphosphoryl)-3'-N-L-phenylalanyl puromycin aminonucleoside, acts as an affinity label in covalently binding to 23-S RNA in the active centre of peptidyl transferase of Escherichia coli ribosomes [Greenwell, P., Harris, R. J. & Symons. R. H. (1974) Eur. J. Biochem. 49, 539-554]. The sequence of a pentanucleotide fragment of 23-S RNA to which the affinity label is attached has now been determined. In an attempt to determine the nucleotide residue to which the affinity label was attached, a 32Plabelled affinity label was reacted with ribosomal RNA, poly(A), poly(C), poly(G) and poly(U) under the conditions used for affinity labelling of ribosomes. However, no specific reaction occurred, confirming our earlier observation that this required the functional integrity of the ribosome. An alternative approach of affinity labelling ribosomes labelled by incubating E. coli in the presence of [³H]adenosine, [³H]cytidine, [³H]guanosine or [³H]uridine, showed that the affinity label was attached to a CMP residue. Effinity-labelled oligonucleotide fragments were purified for sequence analysis as follows. The free α-amino group of the affinity-label molecule attached to 23-S ribosomal [32P]RNA was coupled to biotin by reaction with the N-hydroxysuccinimide ester of biotin. After digestion of the RNA with either ribonuclease A or T1, the basic protein avidin was added to form the avidin, biotin-affinity-label-oligonucleotide fragment. Stepwise chromatography on phosphocellulose, followed (after dissociation of the avidin) by electrophoresis on DEAE-cellulose at pH 1.9 gave substantial purification of the biotin-affinity-label-oligonucleotide. From the nucleotide composition of the purified ribonuclease A and T1 fragments, the pentanucleotide sequence of G-U-C*-C-Gp was obtained, where C* represents C modified by the affinity label. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with out previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A, 73, 702–706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can he compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subnnit interaction. [ABSTRACT FROM AUTHOR]

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3′ and 5′ termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5′ maturation of 16S rRNA is completely blocked. In contrast, 3′ maturation is essentially unaffected. The 5′ unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5′ end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G. [ABSTRACT FROM AUTHOR]

The subunit association capacity of 30S and 50S ribosomal subunits from Escherichia coli mutants lacking protein S20 or L11 as well as of 50S subunits depleted of L7/L12 was tested by sucrose gradient centrifugation and by a nitrocellulose filtration method based on the protection from hydrolysis with peptidyl-tRNA hydrolase of ribosome-bound AcPhe-tRNA. It was found that the subunits lacking either S20 or L11 display an altered association capacity, while the 50S subunits lacking L7/L12 have normal association behavior. The association of S20-lacking 30S subunits is quantitatively reduced, especially at low Mg2+ concentrations (5–12 mM), and produces loosely interacting particles which dissociate during sucrose gradient centrifugation. The association of L11-lacking 50S subunits is quantitatively near-normal at all Mg2+ concentrations and produces loosely associating particles only at low Mg2+ concentrations (5–8 mM); the mechanism of their association with 30S subunits, however, or the structure of the resulting 30S–50S couples is altered in such a way as to cause the ejection of an AcPhe-tRNA molecule pre-bound to the 30S subunits in response to poly(U). [ABSTRACT FROM AUTHOR]

The locations of proteins BL1, BL6, BL23 and BL29 from Bacillus stearothermophilus have been determined on the ribosomal surface by immunoelectron microscopy. All four proteins were localized in the same region of the 50S subunit as their homologous counterparts from Escherichia coli, indicating that the ribosomal architecture is the same in both species. This finding is of great importance as it allows structural data obtained on ribosomes from either organism to be incorporated into a unique ribosome model. [ABSTRACT FROM AUTHOR]

The mutant strain of Escherichia coli, TP28, synthesises ribosomes by an abnormal pathway and accumulates large quantities of 47S ribonucleoprotein particles. The protein complement of mutant 70S ribosomes is normal but 47S particles contain only traces of proteins L28 and L33 and have a significantly reduced content of four other proteins. The mutation reduces the rates of synthesis of L28 and L33 by about half but other widespread alterations ensue. In particular, ribosomal protein synthesis in the mutant strain becomes less well balanced than in its parent: some proteins, particularly those from promoter-proximal genes, are oversynthesized and their excess then degraded. [ABSTRACT FROM AUTHOR]

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]

Escherichia coli ribosomal protein S1 and its mutant, shorter, form m1-S1 were cleaved at internal methionyl residues to yield, respectively, six and five fragments of M1 ranging from 1000 to 24000. Methods are described to isolate the fragments in pure form. Four of the fragments (designated F2a, F2b, F3 and F4) contain between 86 and 215 amino acids and are therefore as large as other ribosomal proteins. Fragment F2a, derived from the N-terminal region, has previously been shown to contain the major ribosome binding domain of S1 [S. Giorginis and A. R. Subramanian (1980) J. Mot Biol. 1411 393 -408]. Here we show that the RNA binding domain of S1 is essentially contained in F3 derived from the middle region of S1 and carrying the nonreactive - SH group. The reactive a SF! group of SI, whose activity is modified by ligand binding, was localized in F2b, a fragment with little RNA binding capacity. The characteristic RNA binding domain and a weak ribosome binding domain of S1 have previously been localized in the large trypsin-resistent core SI-Fl [T. Suryanarayana and A. It Subramanian(1979) J. Mol. Biol. 127, 41-54]. Together these data indicate that two of the key functional domains of SI are located in two regions of the molecule separated by an open, exposed segment. The present study also revealed that the nonreactive - SM group of S1 becomes reactive in m1-S1 by the loss of the remote C-terminal region in the latter. [ABSTRACT FROM AUTHOR]

Analysis of various RNA species from Escherichia coli. The RNAs were prepared under mild conditions without spreading using the shadow-cast technique. The 16-S RNA looks like a flattened balloon of diameter 18 ± 1.9 nm with a smooth surface and regular contours. In contrast, structural domains can be detected in 23-S RNA; typically a central domain is surrounded by five to seven satellite domains, with an overall diameter of 33 ±_ 4.5 nm. Different 23-S RNA preparations, which also behave differently in reconstitution assays, differ with respect to extent and height of the satellite structure. 5-S RNA shows an extended rod-like structure with a length of 11 ± 1.6 nm. A more corpuscular and prominent structure was observed for tRNA, with diameters Phe of 7.7 ± 1.1 nm and 8.9 ± 1.0 nm in case of tRNAPhe, and N-acetylphenylalanyl-tRNAPhe respectively. Analysis of the assembly in vitro of the ribosomal 50-S subunit. The assembly of the 50-S subunit in vitro occurs via three reconstitution intermediates, which are formed subsequently during the first incubation of the two-step reconstitution procedure [Dohme, F. and Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. A total reconstitution was performed, aliquots were withdrawn during the first incubation at various times and analyzed in the electron microscope. A second aliquot was taken at the same times, subjected to a standard second incubation and tested for poly-(Phe)-synthesizing activity in the presence of 30-S subunits. The activity obtained is a measure of the amount of (second plus third) reconsitution intermediates present in the aliquot. With this parameter it was possible to select a series of subsequent assembly states from the electron micrographs which cinematographically illustrate the assembly of the 50-S subunit in vitro. The results indicate that the reconstitution intermediate stage RI[IPQ]50(1) comprises various particles, which evidently are formed consecutively. In contrast, the following reconstitution intermediate stage RI50(2) appears as a homogeneous population of particles. [ABSTRACT FROM AUTHOR]

fMet-tRNAfMet binds reversibly to the donor site (P-site) of Escherichia coli ribosomes both in the absence of messenger and in the presence of ApUpGp and some other oligonucleotides or poly(U). Kinetics of interaction conforms the second-order law. The equilibrium constants and the rate constants of binding are estimated at 0°C. Not only the cognate trinucleotide ApUpGp but also some other oligonucleotides and even poly(U) stimulate the binding. The presence of total deacylated tRNA considerably increases the selectivity of association. [ABSTRACT FROM AUTHOR]

6′-N-[14C]Acetyl-tobramycin and [³H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. The 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6′-N-acetyl-tobramycin. The binding of [³H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6′-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6′-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6′-amino group. [ABSTRACT FROM AUTHOR]

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5′ end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S,A. 74, 5509–5513]. Translation can start at either site using AcPhe-tRNA or fMet-tRNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5′ non-coding region of the messenger. Removal of the 5′-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E, coli ribosomes recognize with high specificity an extra initiation site close to the 5′ extremity of the messenger. The relation- ship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed. [ABSTRACT FROM AUTHOR]

The poly(U)-dependent binding of Tyr-tRNATyr to Escherichia coli ribosomes has been studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNATyr/ribosome), and requires the presence of elongation factor (EF) T (a mixture of EF-Ts and EF-Tu), GTP, NH4+ ions and an aminoglycoside antibiotic (streptomycin, neomycin B, kanamycip B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, one molecule of GTP is hydrolyzed per molecule of Tyr-tRNATyr bound, suggesting that ‘proof-reading’ mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNATyr apparently takes place at the ribosomal acceptor site, since peptide bonds are readily formed with N-acetyl-Phe-tRNA prebound to the ribosomal donor site. In contrast to Phe-tRNAPhe binding, Tyr-tRNATyr binding is impaired by the omission of the 50-S subunit, the replacement of GTP by its non-hydrolyzable analogs guanyl-5′-yl methylene diphosphonate and guanyl-5′-yl iminodiphosphonate, and also by the presence of the antibiotic streptogramin A. This suggests that the correct interaction of Tyr-tRNATyr with the peptidyl transferase centre is essential for the stability of this ligand on the ribosome. Moreover, the aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNATyr on the ribosome. [ABSTRACT FROM AUTHOR]

A method is described for the isolation of highly purified proteins from the 50-S subunit of Escherichia coli ribosomes. All the proteins from the large subunit could be isolated with the exception of L14, L26, L31 and L34. The isolated proteins are functionally active in reconstituted particles. The method consists of successive NH4Cl/EtOH and LiCl washing steps, which split off distinct groups of proteins from the ribosome. The protein groups are further separated by a combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (carboxymethylcellulose) in the presence of 6 M urea, at neutral pH and 4°C. The purity of the proteins was analyzed by two-dimensional gel electrophoresis. In addition, ten protein complexes were isolated and identified. [ABSTRACT FROM AUTHOR]

The metabolism of guanosine 3′,5′-bis(diphosphate) (ppGpp) has been studied in a number of different microbial species including Escherichia coli, Bacillus stearothermophilus, Bacillus subtilis and Bacillus brevis. The results can be summarized as follows. 1. Generally, in the E. coli and B. subtilis systems, a stringent factor catalyses the ppGpp-synthesizing reaction only when activated by uncharged tRNA codon-specifically bound to ribosomes. In the B. stearothermophilus and B. brevis systems the stringent factor does not require activation by ribosomes. The physiological significance of this is reflected by the subcellular distribution: stringent factors requiring ribosomes for activity are exclusively found in ribosomal fractions, while those functioning independently of ribosomes are preferentially present in the high-speed supernatant fraction. Formation of ppGpp is quite specific in that neither ATP as pyrophosphate donor nor GDP (or GTP) as acceptor can be replaced by other nucleotides. However some nucleo- tides, particularly ppApp, effectively inhibit ppGpp production, while others, e.g. ppGpp or A(5′)p4(5′)A, are not inhibitory. 2. Degradation of ppGpp is catalyzed by a guanosine 3′,5′-bis(diphosphate) 3′-pyrophospho-hydrolase present in the ribosomal fraction which specifically releases pyrophosphate from the 3′ position of ppGpp. The activity is apparently associated with ribosomes and, unlike stringent factor, is not completely released under low-ionic-strength conditions. The enzyme has a molecular weight of 65000–70000 and forms dimers and tetramers having molecular weights of 130000 and 260000, respectively. It can also be released from ribosomes by 1 M potassium acetate extraction but then is insoluble in buffers of low ionic strength except in the presence of urea. It is inhibited by tRNA regardless of whether or not ribosomes are present. [ABSTRACT FROM AUTHOR]

It was shown that at 20 mM Mg2+ and 0 or 20 °C each 70-S ribosome binds non-enzymatically about one Phe-tRNAPhe molecule in the A site if poly(U) is used as messenger. Only a minor amount of ribosomes (never exceeding 15%) and after long incubation binds a second molecule of Phe-tRNAPhe and forms diphenylalanyl-tRNA. If the Phe-tRNAPhe preparation contains nonspecific deacylated tRNA, the latter is always bound in the P site up to one molecule per 70-S ribosome. The association constant for this non-specific tRNA binding is equal to (1.4 ± 0.3) × 107 M-1 and is independent of the messenger poly(U) and the filling of the A site by a specific aminoacyl-tRNA. It was found that the codon-dependent binding of Phe-tRNAPhe in the A site is reversible, but two different types of complexes are formed with drastic differences of binding constants. This heterogeneity is due to the presence of two conformers of Phe-tRNAPhe. The equilibrium of the two Phe-tRNAPhe conformers depends on Mg2+ and can be shifted if tRNA is heated and Mg2+ added or sequestered by complexing agents. One type of Phe-tRNAPhe yields complexes with 70-S ribosomes showing extremely high affinity constants (higher than 1011 M-1). The second conformer is characterized by a comparatively low binding constant about 5 × 106 M-1. In the binding of both species to 30-S subunits no difference is seen. A big difference in the behaviour of both Phe-tRNAPhe conformers is found also in the absence of messenger RNA. The association constant of Phe-tRNAPhe (high-affinity conformer) to 70-S ribosomes is about 3 × 108 M-1, but for the low-affinity conformer is hundred times lower. [ABSTRACT FROM AUTHOR]

Polysomes containing nascent chains of alkaline phosphatase have been isolated from a membrane-bound polysome preparation. Indirect immunoprecipitation using conformation-specific antibodies has been employed. This technique provides a good enrichment of these polyribosomes since routinely no more than 10–15% of non-specific immunoprecipitation was observed. The yield of the procedure is generally 40% but can be increased if higher non-specific immunoprecipitation is tolerated. Antibodies, previously described, directed against uncoiled or folded monomers of alkaline phosphatase can be used as primary antibody to recognize the nascent chains contained in membrane-bound polysomes which suggests that these chains are partially folded. [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]

Upon digestion of the complex formed from the 23-S ribosomal RNA and the 50-S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered: these fragments contained RNA sections belonging to the 480 nucleotides at the 5' end of 23-S RNA. By determining the sequence of 70 % of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease. Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5' end of 23-S RNA has a compact tertiary structure, which is stabitised by protein L24. [ABSTRACT FROM AUTHOR]

A high-molecular-weight, acidic protein whose amount per ribosomal particle depends on the growth cycle is shown to be associated with Escherichia coli ribosomes. The protein is associated with ribosomes from cells harvested at different phases of the growth cycle, hut a large increase in its amount is seen with ribosomes from post-exponential phase cells. The protein is only slightly washed off the ribosomes by 1 M NH4Cl. When ribosomes are dissociated it remains entirely with the 30-S subunits. We have purified the protein to homogeneity. It has a molecular weight of 70000 and its amino acid composition showed some resemblance to that of ribosomal protein S1. However, the two proteins are shown to be different. These results, together with the earlier-shown variation in the acetylation of L12 during the growth cycle, indicate that certain proteins of (or associated with) E. coli ribosomes may carry specific biochemical roles connected with cellular adaption toward the stationary phase. [ABSTRACT FROM AUTHOR]

A cleavable bifunctional reagent, dimethyl 3,3′-dithiobispropionimidate, has been used to crosslink proteins that occupy neighboring positions in the 30-S ribosomal subunit of Escherichia coli. The crosslinked proteins were identified, fully or partly, by their positions in two two-dimensional gel electrophoretic systems, one diagonal and the other quasi-diagonal, in which the complexes were cleaved after the first-dimensional run. It was found to be necessary to block the protein sulfhydryl groups in order to prevent artifactual disulfide crosslinking after extraction of the protein from the ribosome. Eleven crosslinked complexes were detected. Four were fully identified: the triplet S4-S5-S8, and the pairs s2-S3, S4-S5, and S5-S8. In five others one component was identified unambiguously. No additional complexes were seen when the longer homologous butyro and capro reagents were used. [ABSTRACT FROM AUTHOR]

The dissociation and association behavior of 70-S ribosomes of Escherichia coli has been studied. It has been shown that the dissociation-association reaction can be both a real dynamic equilibrium and a non-equilibrium reaction, dependent upon the ionic conditions of the solvent. At relatively high ionic strength (I = 0.15 M or more) the dissociation-association reaction is an equilibrium reaction, whereas at lower ionic strength (I = 0.1 M or less) there is no dynamic equilibrium between 70-S ribosomes and its subunits. In the latter case a hysteresis in the dissociation-association reaction is observed. [ABSTRACT FROM AUTHOR]

The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequence obtained were: Bacillus stearothermophilus, -G(Z)≈5 Y-U-C-C-U-U-U-C-U (A); B subtilis, -G(Z)≈7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aeruginosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacgteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B. stearothermophilus and B. subtilis, -Y-C; C. cresentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis. [ABSTRACT FROM AUTHOR]

A new labelling technique in vivo has been introduced which allows the tritiation of cell components with high specific activity during growth in rich medium. By this technique the pool size of each protein can be measured directly in the supernatant from centrifugation at 150 000 × g. A measurable pool was found for the proteins S1, S2, S6, S10, L1, L4, L7, L8/9, L10, L12, L21, and L25. Experiments on migration of ribosomal proteins from the supernatant to ribosome (i.e. association) and vice versa (dissociation) demonstrate a remarkable constancy in the composition of the ribosome. There is no significant difference between ribosomes engaged or not engaged in poly-(Phe) synthesis. [ABSTRACT FROM AUTHOR]

16-S RNA and five pure ribosomal proteins (S3, S4, S6, S7 and S8) from Escherichia coli 30-S subunits were studied by circular dichroism in regard to the reconstitution of biologically active subunits. A small structural variation upon heating could be measured for RNA. It cannot be interpreted in terms of stacking or disruption of hydrogen bonds. However, most proteins underwent important structural modifications when heated to 42 °C in reconstitution solvent. This effect was not dependent on the presence of magnesium salt. The relative amounts of secondary structure of native and heat-denatured ribosomal proteins were calculated by using three reference curves obtained from standard proteins, the exact secondary structure of which has been determined by X-ray crystallography. In contrast with earlier results, about 18 %, β-structure was detected in native proteins. This discrepancy can most probably be attributed to the calculation method used. Results are discussed in relation to reconstitution of ribosomes. [ABSTRACT FROM AUTHOR]

Application of the two-dimensional polyacrylamide gel electrophoretic procedure recently described by Mets and Bogorad [4] to the resolution of Escherichia coli ribosomal proteins is described. This procedure is fast (running time of 8-10 h) and highly sensitive (1-2 µg protein per spot) and it gave good resolution for the majority of the 55 proteins in E. coli ribosomes. Moreover, the method has permitted the resolution of a large number of high-molecular-weight. proteins associated with ribosomes not exposed to high-salt washing. Using purified proteins, the positions in the electrophrogram of the individual proteins of the 30-S subunit have been determined. [ABSTRACT FROM AUTHOR]

Phosphatidylserine synthetase occurs in a soluble and a ribosome-bound form in Escherichia coli. The soluble enzyme of E. coli B was partially purified by DEAE-Sephadex chromatography. The ribosomal enzyme was obtained by differential centrifugation. Two types of the enzyme had the same Km (150 μ M) for L-serine. The soluble enzyme was required for the biosynthesis of phosphatidylethanolamine from phosphatidic acid via (CDP-diglyceride in the membrane. The ribosomal enzyme was less effective to the phosphatidylethanolamine formation than the soluble enzyme. By the sucrose-density-gradient centrifugation method the soluble enzyme was proved to be loosely associated with the membrane. [ABSTRACT FROM AUTHOR]

Association between ribosomal subunits of Eschrichia coli can lead to the formation of two classes of 30-S · 50-S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so-called derived subunits. From cross experiments it is concluded that it is the 50-S subunit which determines the nature of the association product, whereas the 30-S subunit determines whether an association product can be formed. Native and derived 50-S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30-8 subunits lose a factor during isolation, probably IF-3. They are therefore able to associate both at 0° and 37 °C. On the other hand derived 30-S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+. A number of trivial factors have been excluded to explain the difference between the two types of 50-S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl-tRNA, known to influence subunit association. The content of tRNA and aminoacyl-tRNA has been determined quantitatively by polyacrylamide-gel electrophoresis. None of the subunits nor the 70-S ribosomes from which the derived subunits are prepared contain a tRNA species. [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]

Poly U-directed stimulation of preincubated calf lens ribosomes revealed that these particles from a mammalian source have a phenylalanine incorporation ability comparable to the Escherichia coli system. A search was made for optimal conditions. After variation of the magnesium concentration during preincubation two levels of maximal incorporation were observed. The region of highest relative stimulation did not coincide with these maxima. Preincubation under conditions of optimal amino acid incorporation yields 80S monomers and to a lesser extent 120S particles. No smaller units or breakdown products were observed in the analytical ultracentrifuge. Such particles could be demonstrated after prolonged dialysis of either polysomes or 80S monomers against 0.1 mM MgC12. Their sedimentation coefficients were 50S and 30S respectively but not 60 and 40 S. [ABSTRACT FROM AUTHOR]

Polyribosomes containing mRNA differentially labelled with [³H]uracil and [14C]uracil in the 5′-and the 3′-proximal portions were isolated from Escherichia coli cells, mRNA degradation to alcohol-soluble products occurred when polyribosomes were incubated in conditions of protein synthesis and proceeded in the 5′ to 3′-direction. 15% of the mRNA was degraded at the end of a 10-min period of incubation. The mRNA-degrading enzyme (or enzymic system) was found to be bound to polyribosomes; it could be removed by washing in low salt. The content in RNAase activity per ribosomes in polyribosomes increased three to five-fold when polyribosomes were isolated from cells in which extensive run-off of ribosomes from mRNA had been induced. On the contrary the RNAase activity in free ribosomes was not affected. This is taken as an indication that the enzyme(s) responsible for the degradation of endogenous mRNA in vitro is not aspecifically adsorbed to ribosomes, but specifically located towards the 5′-end of the mRNA chains in polyribosomes. [ABSTRACT FROM AUTHOR]

A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association-dissociation reaction and of several factors which influence it: the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was -17.4 kcal/mol. Association was accompanied by the net binding of magnesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50-S subunit was dependent on the presence of potassium ions, whereas the change in the 30-S subunit was magnesium-ion dependent. The conformational change in the 50-S subunit was critical for the association-dissociation reaction. Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70-S ribosome. This was an independent reaction which did not influence the subunit association-dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol. [ABSTRACT FROM AUTHOR]