Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis

On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed. There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid. Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U] in the incubation mixture. Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA. The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs. These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis. The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process. A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples.     

Relaxation kinetics of E. coli ribosomes: evidence for the reaction of 30S . IF3 complex with 50S ribosomal subunits.

Addition of initiation factor IF3 to solutions of E. coli ribosomes dramatically alters their behavior in pressure-jump relaxation kinetic experiments in which 90 degrees light-scattering is used to monitor the macromolecular reaction. The effect of IF3 on relaxation processes attributed to "tight" couples is strongly dependent on the Mg2+ concentration. At 2.5 mM Mg2+, addition of 1 molar equivalent of IF3 decreases the relaxation amplitude by a factor of 3 relative to ribosome solutions without IF3. However, at 5.0 mM Mg2+, addition of 1 molar equivalent of IF3 produces a marked increase in the relaxation amplitude, by a factor of 2-8 fold relative to ribosomes in the absence of IF3. IF3 has no effect on the relaxation process attributed to "loose" couples at 10 mM Mg2+. While we are unable to propose a precise mechanism for IF3 action with the data on hand, our results require that the 30S . IF3 complex either reacts with the 50S subunit, forming a 70S . IF3 intermediate, or acts as a pool of reactive 30S subunit. Further kinetic evidence is required to distinguish between these possible pathways.     

Relaxation kinetics of E coli ribosomes: evidence for the reaction of 30S . IF3 complex with 50S ribosomal subunits

Addition of initiation factor IF3 to solutions of E.coli ribosomes dramatically alters their behavior in pressure-jump relaxation kinetic experiments in which 90 degrees light-scattering is used to monitor the macromolecular reaction. The effect of IF3 on relaxation processes attributed to "tight" couples is strongly dependent on the Mg2+ concentration. At 2.5 mM Mg2+, addition of 1 molar equivalent of IF3 decreases the relaxation amplitude by a factor of 3 relative to ribosome solutions without IF3. However, at 5.0 mM Mg2+, addition of 1 molar equivalent of IF3 produces a marked increase in the relaxation amplitude, by a factor of 2-8 fold relative to ribosomes in the absence of IF3. IF3 has no effect on the relaxation process attributed to "loose" couples at 10 mM Mg2+. While we are unable to propose a precise mechanism for IF3 action with the data on hand, our results require that the 30S . IF3 complex either reacts with the 50S subunit, forming a 70S . IF3 intermediate, or acts as a pool of reactive 30S subunit. Further kinetic evidence is required to distinguish between these possible pathways.     

Differences in physical and biological properties of 50S ribosomes and 23S RNAs derived from tight and loose couple 70S ribosomes

Tight couple (TC) 50S ribosomes on treatment with kethoxal lose their capacity to associate with 30S ribosomes whereas loose couple (LC) 50S ribosomes on such treatment fully retain their association capacity. The same is true for 23S RNAs isolated from treated 50S ribosomes or isolated 23S RNAs directly treated with kethoxal, so far as their capacity to associate with 16S RNA is concerned. At certain Mg++ concentrations TC 23S RNA is highly susceptible to the nucleolytic action of single-strand specific enzyme RNase I; LC 23S RNA is quite resistant. The Mg++-dependencies of the two species of 23S RNAs for association with 16S RNA are also quite different. The fluorescence enhancement of ethidium bromide due to binding to TC 23S RNA is slightly less than LC 23S RNA. The hyperchromicity of LC 23S RNA due to thermal denaturation is somewhat more than TC 23S RNA. LC 23S RNA has slightly more elliptic CD spectrum than TC 23S RNA. These results clearly show that 23S RNAs present in TC and LC 50S ribosomes are distinct from each other. It has been recently demonstrated in this laboratory that they can be interconverted by the agents involved in translocation and thus appear to be conformomers.     

Structural dynamics of bacterial ribosomes: II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA

Ribosomes from Escherichia coli were tested for activity in initiation with R17 RNA as messenger. All vacant 70 S ribosomes but not all subunits were found to be active. The ability of 30 S and 50 S subunits to form a 70 S couple at Mg²⁺ concentrations above 4 mm is a stringent test for activity.Fresh extracts, prepared at 10 mm-Mg²⁺ from cells harvested after slow cooling contain up to 80% of the ribosomes in the form of vacant 70 S couples and 20% of free subunits. The proportion of subunits increases with standing as a result of the preferential inactivation of the 50 S particles. “Native” subunits are heterogeneous and consist mostly of active 30 S and inactive 50 S particles.In contrast to 50 S subunits, 30 S subunits prepared by exposure of 70 S ribosomes to low Mg²⁺ concentrations, are largely inactive and unable to reassociate with their active 50 S counterparts. However, both initiation and association activity can be restored by heating.The results imply that the structures necessary for subunit association are most critical for the biological activity of ribosomes, presumably because they are topologically closely related to the binding sites for messenger RNA, transfer RNA, and the protein factors for initiation, translocation and termination.     

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

A Comparison of the Native and Derived 30S and 50S Ribosomes of Escherichia coli

Four types of ribosomes occurring in E. coli have been separated by sucrose gradient centrifugation. These are the 30S and 50S particles occurring in E. coli extracts (native particles), and the 30S and 50S particles which are the subunits of 70S ribosomes (derived particles). Two criteria were used in comparing these particles: (1) The type of RNA contained in each, as determined by sedimentation velocity in the analytical ultracentrifuge. (2) The ability of mixtures of 30S and 50S ribosomes (derived 30S + derived 50S, native 30S + native 50S) to undergo the reaction: [Formula: see text] Native and derived 30S particles were found to contain 16S RNA. Derived 50S particles contained 23S RNA and a small amount of 15 to 20S RNA, whereas native 50S ribosomes contained only 16S RNA. Derived 30S and 50S particles combined to form 70S particles. However, under identical conditions, native 30S and 50S particles did not form 70S ribosomes.     

Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.     

Structural study of translating 70 S ribosomes from Escherichia coli: I. Electron microscopy

Translating 70 S ribosomes of Escherichia coli either in the pre-translocation or in the post-translocation state have been prepared by using the cell-free translation system in poly(U)—S—S—Sepharose columns [Methods Enzymol. (1979) 59, 382–398]. Electron microscopy study of the preparations has demonstrated that: (1) the mutual orientation of the ribosomal subunits in the translating ribosomes is the same as proposed by Lake for routine 30 S·50 S couples [J. Mol. Biol. (1976) 105, 111–130]; (2) the L7/L12 stalk of the 50 S subunit sticks out from the 70 S particle and does not join the 30 S subunit; (3) pre-translocation and post-translocation state ribosomes do not differ in mutual orientation of the subunits and in the position of the L7/L12 stalk, within the limits of electron microscopy resolution.     

Sucrose density gradient sedimentation of E. coli ribosomes.

The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg²⁺.     

Structural dynamics of bacterial ribosomes: IV. Classification of ribosomes by subunit interaction

Previous studies in this series (M. Noll et al., 1973a,b; Noll & Noll, 1974) have established that in Escherichia coli the ability of subunits to form vacant 70 S ribosome couples at 10 mm-Mg²⁺ is a stringent condition for activity in the translation of natural messenger (R17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into two classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mm-Mg²⁺ and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mm-Mg²⁺ in the gradient, both species sediment as a 70S peak. At 10 mm-Mg²⁺ and 60,000 revs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples.Association is dependent on the state of each subunit. Removal of Mg²⁺ produces 30 S b-particles that are unable to associate with 50 S subunits unless reconverted to the 30 S a-form by thermal activation according to Zamir et al. (1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a → 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with RNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association step.Fresh S-30 extracts contain only tight couples (approx. 80%) and subunits (approx. 20%). Our results suggest that loose couples are artefacts derived from tight couples by a structural or conformational modification.Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA_Phe (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization, are shown to consist of the b-form of each subunit.It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due to the lack of structural criteria required for standardizing the ribosome preparation used by different investigators. This study provides simple methods and criteria to classify and separate physically all ribosome and ribosome subunits that have been observed into well-defined classes of predictable activity.     

Polyclonal antibodies as probes to distinguish between tight and loose couple 50S ribosomes of Escherichia coli.

Antibody has been raised in rabbit against L7/L12 protein of E. coli 50S ribosomes and purified, finally through affinity column. A sensitive assay method using ELISA technique has also been standardised. LC 50S ribosomes react more with the antibody than TC 50S ribosomes. This supports the earlier physical data [Burma D P, Srivastava A K, Srivastava S, Tewari D S, Dash D & Sengupta S K, (1984), Biochem Biophys Res Commun, 124, 970] indicating that L7/L12 stalk region is protruded in medium in LC ribosomes and folded towards the body in TC ribosomes.     

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.     

Composition of the Escherichia coli 70S ribosomal interface: a cross-linking study

70S tight-couple ribosomes from Escherichia coli were cross-linked by using the bifunctional reagent phenyl-diglyoxal (PDG). The reaction was stopped after 4-h incubation while still in the linear range. In comparison with untreated ribosomes, 30% of those treated with PDG were shown, by sucrose gradient experiments, not to be separable into their subunits, but remained as 70S particles. There was no detectable change in the structure of the reacted particles when their sedimentation behavior was compared with that of native 70S controls. When the cross-linking reaction was performed in the presence of tRNAPhe and poly(U), the reacted ribosomes retained 40-50% of their tRNA binding activity. The reaction leads predominantly to the formation of RNA-protein cross-links but protein--protein as well as RNA-RNA cross-links could also be detected. Cross-linked material was extracted, and the individual RNAs were separated into 23S, 16S, and 5S RNAs. Proteins were identified electrophoretically after reversal of the RNA-protein cross-links. Proteins were found to be cross-linked to RNAs within and across the ribosomal subunits; the latter are considered to be close to or at the 70S subunit interface. The arrangement of RNA and protein at the subunit interface is discussed.     

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.     

Nuclear genome codes for chloroplast ribosomal proteins in Acetabularia. I. Isolation and characterization of chloroplast ribosomal particles

A method is described for the isolation of chloroplast ribosomes from Acetabularia cells in yields sufficient for the characterization of these particles. Ribosomal particles sedimenting with 70S, 56S, 44S, and 30S have been obtained. The monoribosome sediments with 70S and dissociates into a larger 44S and a smaller 30S subunit. The sedimentation behaviour of the particles as well as the equilibrium between monoribosomes and their subunits is not influenced by the centrifugation step as could be revealed by formaldehyde fixation.     

Probing ribosome function and the location of Escherichia coli ribosomal protein L5 with a monoclonal antibody

A monoclonal antibody specific for Escherichia coli ribosomal protein L5 was isolated from a cell line obtained from Dr. David Schlessinger. Its unique specificity for L5 was confirmed by one- and two-dimensional electrophoresis and immunoblotting. The antibody recognized L5 both in 50 S subunits and 70 S ribosomes. Both antibody and Fab fragments had similar effects on the ribosome functions tested. Antibody bound to 50 S subunits inhibited their reassociation with 30 S subunits at 10 mM Mg2+ but not 15 mM, the concentration present for in vitro protein synthesis. The 70 S couples were not dissociated by the antibody. The antibody caused inhibition of polyphenylalanine synthesis at molar ratios to 50 S or 70 S particles of 4:1. The major inhibitory effect was on the peptidyltransferase reaction. There was no effect on either elongation factor binding or the associated GTPase activities. The site of antibody binding to 50 S was determined by electron microscopy. Antibody was seen to bind beside the central protuberance or head of the particle, on the side away from the L7/L12 stalk, and on or near the region at which the 50 S subunit interacts with the 30 S subunit. This site of antibody binding is fully consistent with its biochemical effects.     

The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation.

To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A18 mutation in this structure element. Previously, this mutation, which changes the central base pair of helix 2, C18--G917, to an A18xG917 mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested. Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits. Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control. However, the A18 mutation has a profound effect on the overall stability of the subunit. The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins. Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins. Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution. Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins. We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex. We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.