Conformational change of L7/L12 stalk in the different functional states of 50S ribosomes

Conformational change of 50S ribosomes takes place during protein synthesis. The primary change is most likely in the secondary or tertiary structure of rRNA in the L7/L12 stalk region. In order to throw further light on this conformational change, the change in fluorescence of tight couple 50S ribosomes on conversion to loose couple 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-l-sulphonic acid-labelled L7/L12, following the treatment with elongation factor-G and 5′-guanylyl methylene diphosphate was measured. It was enhanced in agreement with the results reported earlier. Further, the quenching of fluorescence of 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-1-sulphonic acid-labelled L7/L12 by acrylamide was studied. The quenching is more in case of loose couples. On conversion of loose couple 50S ribosomes to tight couple ones the quenching becomes less whereas the reverse happens on conversion of tight couple 70S ribosomes to loose couples. These results indicate the conformational change of L7/L12 stalk in the different functional states of 50S ribosomes.     

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.     

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.     

Ribosomal activity of the 16 S.23 S RNA complex

It has been demonstrated in this laboratory that 16 S and 23 S RNAs form a binary complex like 30 S and 50 S ribosomes under certain specific conditions, and 5 S RNA can be incorporated into the complex in stoichiometric amounts in presence of three ribosomal proteins, L5, L18, and L15/25. These studies raised the basic question of whether such complex will have biological activity. Therefore, the following steps in protein synthesis were examined with the complex in place of the ribosomes: (i) poly-U-dependent binding of phenylalanyl tRNA; (ii) EF-G-dependent GTPase activity; (iii) initiation complex formation; (iv) peptidyl transferase activity; and (v) poly-U-dependent polyphenylalanine synthesis. All the steps could be unequivocally demonstrated by the addition of a limited number of proteins although the complex had comparatively much less activity than 70 S ribosomes. It appears that rRNAs are directly involved in various steps of protein synthesis. Furthermore, the 16 S.23 S RNA complex might have acted as a primitive ribosome, as suggested by Crick and Orgel.     

Do ribosomal RNAs act merely as scaffold for ribosomal proteins?

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the Binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-Binding, (B) poly U-dependent Binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has Been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (P) site. A model has Been proposed for translocation on the Basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to Be noncontroversial.     

Pressure-induced dissociation of tight couple ribosomes

Ribosomes from Escherichia coli have been shown to undergo subunit dissociation at elevated hydrostatic pressure. This holds for both crude and highly purified ribosomes. No inhibitory effect could be detected by addition of either the S100 supernatant, or tRNA, polyuridylic acid, and spermine. Light scattering experiments at pressures up to 1000 bar reveal different susceptibility of tight couple and loose couple ribosomes toward pressure dissociation. Tight couples are subjected to EF-Tu-catalyzed binding of aminoacyl-tRNA, thus yielding a model system of the elongating ribosome before the peptidyl transfer step. High pressure dissociation of this compound suggests that enzymatic binding converts tight couples into loose couples. A hypothesis referring to conformational changes during the elongation cycle is presented.     

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg²⁺ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.     

Conformational change of 50 S ribosomes on enzymatic binding of phenylalanyl-tRNA

Tight couple 70 S ribosomes are converted to loose couple ones on enzymatic binding of phenylalanyl-tRNA. Enzymatic binding at 0 degree C as well as nonenzymatic binding does not lead to any change. Further, no change takes place when the P site is occupied by N-acetylphenylalanyl-tRNA. Loose couple 70 S ribosomes are not affected by either enzymatic or nonenzymatic binding of phenylalanyl-tRNA.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.     

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.     

Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine

5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N¹-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA^Phe at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent ‘loosening’ of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.     

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & St?ffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].     

Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors

Alpha-sarcin cleaves one phosphodiester bond of 23S rRNA within 70S ribosomes or 50S subunits derived from E. coli. The resulting fragment was isolated and sequenced. The cleavage site was identified as being after G2661 and is located within a universally conserved dodecamer. Cleavage after G2661 specifically blocked the binding of both elongation factors, i.e. that of the ternary complex Phe-tRNA*EF-Tu*GMPPNP and of EF-G*GMPPNP, whereas all elongation-factor independent functions of the ribosome, such as association of the ribosomal subunits, tRNA binding to A and P sites, the accuracy of tRNA selection at both sites, the peptidyl transferase activity, and the EF-G independent, spontaneous translocation, were not affected at all. Control experiments with wheat germ ribosomes yielded an equivalent inhibition pattern. The data suggest that the universally conserved dodecamer containing the cleavage site G2661 is located at the presumably overlapping region of the binding sites of both elongation factors.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Relaxation kinetics of E. coli ribosomes.

Relaxation kinetics measurements on two types of ribosome preparations were parformed by the pressure-jump and temperature-Jump techniques, using light scattered at 90° as detector. For freshly prepared tibosomes isolated as 70S tight coupled from 26 000 RPM sucrose gradint sedimentation in 10 mM Mg²⁺, surprisingly large reaction amplitudes were found in 10 mM Mg²⁺ wilh both techniques, leading to an overall formation constant for 70S couples approximately three orders of magnitude smaller than that reported fot tight couples. For pelleted, two-tunes salt-washed ribosomes, amplitude titration versus Mg²⁺ in the pressure-jump apparatus showed an amplitude maximum near 10 mM Mg²⁺ with a relaxation time near 20 ms, and a second amplitude maximum near 2.5 mM Mg²⁺ with a relaxation time near 25 s. Both types of preparation on reanalysis on sucrose gradients at 5 mM Mg²⁺ showed approximately 15% of subunits, with a distinct zone in the 50S region. 70S light couples recovered from a sucrose density gradient separation at 5 mM Mg²⁺ on pelleted two-times salt-washed ribosomes behaved in the same way as the original sample in pressure-jump experiments at 10 mM Mg²⁺. These findings have been interpreted as follows (I) the processes observed at 10 mM Mg²⁺ are due entirety to the relatively small loose couple content of the samples, even in the case of material isolated as 70S tight couples, (2) the processes observed at 2.5 mM Mg²⁺ are due almost entirely to the preponderant tight couple population of the material, and (3) samples isolated as 70S tight couples from sucrose gradients at 5 mM Mg²⁺ spontaneously revert within hours into micro-heterogeneous material containing about 15% loose couples, for both types of ribosomes.     

The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli

During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.     

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.     

The distance between two functionally significant regions of the 50 S Escherichia coli ribosome: the erythromycin binding site and proteins L7L12

The distance between the erythromycin binding site on the 50 S Escherichia coli ribosome and protein L7 has been measured by singlet-singlet energy transfer. A non-covalently bound erythromycin derivative, fluoroscein isothiocyanate erythromycylamine, was used as the acceptor. This derivative can be completely displaced from ribosomes by erythromycin, suggesting that they have the same binding site. 1,5-Iodoacetylethylenediamine naptholsulfonate-labeled protein L7 served as the fluorescent donor. It was reconstituted with salt/ethanol-washed 50 S cores. This readdition was accompanied by total recovery of elongation factor G-dependent GTPase activity. This suggests that the protein modification does not significantly perturb 50 S function or structure. Energy transfer measurements by both static and lifetime techniques were in good agreement. After consideration of various errors that enter the measurements and calculations, the L7-erythromycin distance is estimated to be 70 ± 10 Å. This long distance is interesting, since both sites may be involved in translocation.The fluorescent derivative of erythromycin was also used to study binding kinetics to the 50 S and 70 S ribosomes. Binding is a simple second-order step and proceeds about 11 times faster on the 70 S particle. Exchange of the fluorescent derivative with excess erythromycin is limited by the dissociation rate, and this is four times faster on the 70 S particle. These results suggest that the erythromycin site is more accessible on the 70 S particle, and may be an indication of conformational changes in the 50 S ribosome upon combination with the 30 S ribosome.     

Structural diversity in bacterial ribosomes: mycobacterial 70S ribosome structure reveals novel features

Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria.