Ribosomal complexes from an extremely halophilic bacterium and the role of cations

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.     

A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.     

Antagonistic action between spermidine and putrescine on association and dissociation of purified, run-off ribosomes from Escherichia coli.

The effects of polyamines on the equilibrium between prokaryotic ribosomal subunits and 70 S ribosomes have been studied as a function of concentration of Mg2+ from 2.5 to 7.5 mM. Run-off ribosomes were obtained from Escherichia coli and were washed with buffered 1 M NH4C1. Spermidine at 1 mm favors association of subunits at all concentrations of Mg2+. Putrescine, at concentrations above 8 mM, favors net dissociation at concentrations of Mg2+ below 4.5 mM. Streptomycin behaves like spermidine, while putrescine behaves like initiation factor 1 and initiation factor 3. The effect of putrescine on dissociation is time-dependent and appears to have a half-life of about 3.5 min at 30 degrees. When added after the effects of spermidine or streptomycin on association have occurred, putrescine still causes dissociation. The data suggests that putrescine may reduce net formation of vacant 70 S ribosomes. Another possibility is that putrescine and spermidine may act antagonistically to maintain a labile equilibrium between ribosomal subunits and vacant 70 S ribosomes. It may be significant that the putrescine effect is observed at the concentration of Mg2+ found to be optimum for initiation.     

Mechanism of the ribosome-dependent uncoupled GTPase reaction catalyzed by polypeptide chain elongation factor G.

At low NH4-+ concentrations, 50S ribosomal subunits from E. coli were fully active in the absence of 30S ribosomal subunits, in forming a complex with the polypeptide chain elongation factor G (EF-G) and guanine nucleotide (ternary complex formation), and also in supporting EF-G dependent hydrolysis of GTP (uncoupled GTPase reaction). However, both activities were markedly inhibited on increasing the concentration of the monovalent cation, and at 160 mM NH4-+, the optimal concentration for polypeptide synthesis in a cell-free system, almost no activity was observed with 50S ribosomes alone. It was found that the inhibitory effect of NH4-+ was reversed by addition of 30S subunits. Thus, at 160 mM NH4-+, only 70S ribosomes were active in supporting the above two EF-G dependent reactions, whereas at 20 mM NH4-+, 50S ribosomes were almost as active as 70S ribosomes. Kinetic studies on inhibition by NH4-+ of the formation of 50S ribosome-EF-G-guanine nucleotide complex, indicated that the inhibition was due to reduction in the number of active 50S ribosomes which were capable of interacting with EF-G and GTP at higher concentrations of NH4-+. The inhibitory effects of NH4-+ on ternary complex formation and the uncoupled GTPase reaction were markedly influenced by temperature, and were much greater at 0 degrees than at 30 degrees. A conformational change of 50S subunits through association with 30S subunits is suggested.     

Comparison of active and inactive forms of the E. coli 30S ribosomal subunits

Dissociation of E. Coli 70S ribosomes in the presence of 0.1 mM Mg++ yields partially inactivated 30S and 50S subunits. This inactivation can be avoided by dissociating the 70S ribosome in a medium containing 10 mM Mg++. 400 mM Na+. Comparison of the active and inactive forms of the 30S and 50S subunits has led to the following conclusions: 1) The two forms possess identical (50S subunits) or very similar (30S subunits) hydrodynamic properties. No differences in their morphologies is detectable by electron microscopy. 2) They possess the same protein compositions except for the presence of a larger amount of protein S1 in the inactive than in the active form of the 30S subunit. 3) They differ significantly in functional properties: more efficient association of the active than of the inactive forms with the complementary subunit; extensive dimerization of inactive 30S subunits in the presence of 10 mM Mg++; no dimerization of active 30S subunits under the same conditions; six-fold higher peptidyl transferase activity of active as compared to inactive 50S subunits.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

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

The binding of ribosomal protein S1 to S1-depleted 30S and 70S ribosomes. A fluorescence anisotropy study of the effects of Mg2+

We have determined the equilibrium constants for the binding of AEDANS-labelled S1 to S1-depleted 30S and 70S ribosomes. For "tight" ribosomes, the association of S1 increases with the sixth power of Mg2+ concentration, but for 30S subunits and "loose" ribosomes, there is virtually no dependence of the association on Mg2+ over the same concentration range, 2-10 mM in Mg2+. The binding of S1 to 70S ribosomes at 10 mM Mg2+ is stabilized by 2 kcal/mol compared to the binding to 30S subunits. When intact S1 binds to tight ribosomes, the fluorescence anisotrophy is that for virtually complete rotational immobilization. The anisotropies vary considerably with the preparation and treatment of both S1 and ribosomes and these variations are detailed here. The results suggest the linkage of Mg2+-dependent conformational changes in the intact ribosomes, perhaps including rRNA, and the binding of S1.     

Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit

In vitro mutagenesis of rplB was used to generate changes in a conserved region of Escherichia coli ribosomal protein L2 between Gly221 and His231. Mutants were selected by temperature sensitivity using an inducible expression system. A mutant L2 protein with the deletion of Thr222 to Asp228 was readily distinguishable from wild-type L2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ribosomes from the strain overexpressing this mutant protein were characterized by sucrose density gradient centrifugation and protein composition. In addition to 30 S and 50 S ribosomal subunits, cell lysates contained a new component that sedimented at 40 S in 1 mM Mg2+ and at 48 S in 10 mM Mg2+. These particles contained mutant L2 protein exclusively, completely lacked L16, and had reduced amounts of L28, L33, and L34. They did not reassociate with 30 S ribosomal subunits and were inactive in polyphenylalanine synthesis. Other mutants in the same conserved region, including the substitution of His229 by Gln229, produced similar aberrant 50 S particles that sedimented at 40 S and failed to associate with 30 S subunits.     

Immunoprecipitation of 70S, 50S and 30S ribosomes ofEscherichia coli

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

Study of mammalian ribosomal protein reactivity in situ. I. - Effect of 2-methoxy-5-nitrotropone on 40S and 60S subunits.

Liver ribosomes and subunits were reacted with increasing concentrations of 2-methoxy-5-nitrotropone. At low reagent concentrations (0.3 mM), the molar uptake by 60S subunits was more efficient than the uptake by 40S subunits, and the amount of reagent bound to 80S ribosomes was less than that bound to both free subunits considered together. At higher reagent concentrations, the molar uptake of both subunits was equivalent. Subunits and ribosomes remained fully active when reacted with up to 0.3 mM and 1 mM of the reagent, respectively. With 2 mM of the reagent, both subunits were half inactivated, although their sedimentation characteristics were unaltered. The reactivity of each ribosomal protein was assessed by two-dimensional gel electrophoresis and quantitative measurement of the unmodified proteins. From these results, considered together with the uptake characteristics and the inactivation curves, a number of tentative conclusions about ribosome topography can be drawn. The over-all sensitivity of the 60S subunits to the reagent is higher than that of the 40S subunits. Both subunits undergo a conformational change when they combine to form 80S ribosomes. Proteins S18, S20, S28 and L5, L9, L11, L15, L16, L25, L29, L30, L31, L34, L37 have NH2 groups exposed in native subunits. These groups are not essential for subunit function.     

Formation of 70S ribosomes: large activation energy is required for the adaptation of exclusively the small ribosomal subunit

Association of ribosomal subunits is an essential reaction during the initiation phase of protein synthesis. Optimal conditions for 70S formation in vitro were determined to 20 mM Mg2+ and 30 mM K+. Under these conditions, the association reaction proceeds with first order kinetics, suggesting a conformational change to be the rate-limiting step. 70S formation separates into two sub-reactions, the adaptation of the ribosomal subunits to the association conditions and the association step itself. The activation energy of the process was determined to 78 kJ/mol and revealed to be required exclusively for the adaptation of the small subunit, rather than the large subunit or the association step. The presence of mRNA [poly(U)] together with cognate AcPhe-tRNA, accelerates the association rate significantly, forming a well-defined 70S peak in sucrose gradient profiles. mRNA alone provokes an equivalent acceleration, however, the resulting 70S couple impresses as an ill-defined, broad peak, probably indicating the readiness of the ribosome for tRNA binding, upon which the ribosome flips into a defined state.     

Chain initiation factor 3 crosslinks to E. coli 30S and 50S ribosomal subunits and alters the UV absorbance spectrum of 70S ribosomes

We report a direct procedure to determine the proteins near the IF-3 binding site in purified 30S and 50S ribosomal subunits. This procedure introduces only limited numbers of cleavable crosslinks between IF-3 and its nearest neighbors. The cleavable crosslinking reagent, 2-iminothiolane, was used to crosslink IF-3 in place to both 30S and 50S subunits. Ribosomal proteins S9/S11, S12, L2, L5 and L17 were found, by this approach, to be in close proximity to the factor in purified IF-3-subunit complexes. In addition, IF-3 was shown to alter the ultraviolet absorbance spectrum of E. coli 70S ribosomes at 10 mM Mg2+. The magnitude of the observed difference spectrum at a constant IF-3/ribosome ratio of 1.0, is linearly dependent upon ribosome concentration over the range 5 nM - 55 nM. Titration experiments indicated that the observed effect is maximal at an IF-3/ribosome ratio of approximately 1.0. These results are taken to indicate a conformational change in the 70S ribosome induced by IF-3.     

Stimulation of peptidyltransferase activity of 50S subunits by alcohols

We have demonstrated that in certain conditions 50S subunits can transfer peptide moiety from peptidyl-tRNA to puromycin in the absence of alcohol. Monovalent cations NH4+ and K+ support this reaction, while Na+ and Li+ are ineffective. Optimal concentration for NH4+ is 1.8 M. Mg2+ ion concentrations above 12 mM are needed as well as an elevated temperature (30 degrees C). Using the alcohol-free puromycin reaction of 50S subunits we show that alcohol activates the peptidyl transferase center, but does not participate in the puromycin reaction. Neither does it change the protein composition of subunits.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

Binding of S21 to the 50S subunit and the effect of the 50S subunit on nonradiative energy transfer between the 3' end of 16S RNA and S21

Escherichia coli ribosomal protein S21 was labeled at its single cysteine group with a fluorescent probe. Labeled S21 showed full activity in supporting MS2 RNA-dependent binding of formylmethionyl-tRNAf to 30S ribosomal subunits. Fluorescence anisotropy measurements and direct analysis on glycerol gradients demonstrate conclusively that labeled S21 binds to 50S ribosomal subunits as well as to 30S and 70S particles. The relative binding affinities are in the order 70S greater than 30S greater than 50S. Other results presented appear to indicate that S21 is bound in the same position on either 50S subunits or 30S subunits as in 70S ribosomes, suggesting that the protein is bound simultaneously to both subunits in the latter. Addition of 50S subunits to 30S particles containing probes on S21 and at the 3' end of 16S RNA caused a decrease in the energy transfer between these points. The results correspond to an apparent change in distance from 51 to 61 A.     

Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method

A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.     

A single base change at 726 in 16S rRNA radically alters the pattern of proteins synthesized in vivo.

A single base change in 16S rRNA (C-726 to G) was constructed by site-directed mutagenesis and cloned into the multicopy plasmid pKK3535 (generating pKK726G) which contains the complete rrnB operon from Escherichia coli. The mutant 16S rRNA was found predominantly in the 30S subunit fraction but was present in the 70S ribosomes. Protein analyses of the free 30S subunits revealed a decrease in the levels of ribosomal proteins S2 and S21 while the composition of the 70S ribosomes was as the wild-type. Transformants of pKK726G were temperature sensitive for growth, although the mutant ribosomes themselves were translationally active in vivo at 37 and 42 degrees C. Two-dimensional gel electrophoresis of the proteins translated in vivo revealed an altered protein profile which included novel proteins, changes in the levels of normal proteins, and the presence of heat shock proteins (HSPs) at 30 degrees C. Inactivation of the host encoded wild-type ribosomes coincided with a significant decrease in the synthesis of the HSPs. We therefore believe the induction of the HSPs to be a secondary response by the cells to the presence of the abnormal proteins.     

Probing the functional role and localization of Escherichia coli ribosomal protein L16 with a monoclonal antibody

A monoclonal antibody specific for Escherichia coli ribosomal protein L16 was prepared to test its effects on ribosome function and to locate L16 by immunoelectron microscopy. The antibody recognized L16 in 50 S subunits, but not in 70 S ribosomes. It inhibited association of ribosomal subunits at 10 mM Mg2+, but not at 15 mM Mg2+. Poly(U)-directed polyphenylalanine synthesis and peptidyltransferase activities were completely inhibited when the L16 antibody was bound to 50 S subunits at a molar ratio of 1. There was no inhibitory effect on the binding of elongation factors or on the associated GTPase activities. Fab fragments of the antibody gave the same result as the intact antibody. Chemical modification of the single histidine (His13) by diethyl pyrocarbonate destroyed antibody binding. Electron microscopy of negatively stained antibody subunit complexes showed antibody binding beside the central protuberance of the 50 S particle on the side away from the L7/L12 stalk and on or near the interface between the two subunits. This site of antibody binding is fully consistent with its biochemical effects that indicate that protein L16 is essential for the peptidyltransferase activity activity of protein biosynthesis and is at or near the subunit interface.     

30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15

Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.