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.     

Protection of specific sites in 23 S and 5 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman &; Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are

in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences,

is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G₁₃ is partially protected in 70 S ribosomes. while G₄₁ remains unaffected by subunit association.The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.     

Mechanism of ribosomal subunit association: discrimination of specific sites in 16 S RNA essential for association activity.

Modification of 30 S ribosomal subunits with kethoxal causes loss of their ability to associate with 50 S subunits under tight couple conditions. To identify those 16 S RNA sequences important for the association. ³²P-labeled 30 S subunits were partially inactivated by reaction with kethoxal. The remaining association-competent 30 S subunits were selected from the modified population by their ability to form 70 S ribosomes. Comparison of kethoxal diagonal maps of the association-competent subunits with those of the total population of modified subunits reveals nine sites in 16 S RNA whose modification leads to loss of association activity. Eight of these sites were previously found to be protected from kethoxal attack and one was shown to have enhanced reactivity in 70 S ribosomes (Chapman &; Noller, 1977). As before, these sites are not distributed thoughout the molecule, but are found to be clustered in two regions, at the middle and at the 3′ terminus of the 16 S RNA chain.We interpret these findings in terms of a simple preliminary model for the functional organization of 16 S RNA, supported by the observations of other investigators, in which we divide the molecule into four domains. (1) Residues 1 to 600 are involved mainly in structural organization and assembly. (2) Residues 600 to 850 include sites which make contact with the 50 S subunit and are essential for subunit association. (3) Sites from the domain comprising residues 850 to 1350 line a pocket at the interface between the two ribosomal subunits. and contribute to the binding site(s) for transfer RNA. (4) Residues 1350 to 1541 also contain sequences which bind the 50 S subunit, but some sites in this domain alternatively participate in the initiation of protein synthesis.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

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.     

Mechanism of ribosomal subunit association: oligodeoxynucleotides as probes.

From the kethoxal treatment data [Herr, W.; Chapman, N.M.; Noller, H.F. (1979) J. Mol. Biol. 130, 433-439] some regions of ribosomal RNAs are thought to be responsible for the association of 30S and 50S ribosomes of E. coli to form 70S ribosomes. In order to test this possibility about a dozen oligodeoxynucleotides complementary to the suspected regions of rRNAs were synthesised. Their association with ribosomes and naked rRNAs was tested by the gel filtration technique. In order to check the effects on the ribosomal subunit association or rRNA association either intact 30S and 50S ribosomes or naked 16S and 23S rRNAs were preincubated with the individual oligodeoxynucleotide and its effect was checked by density gradient centrifugation followed by UV absorbance monitoring. Some oligodeoxynucleotides interfered with either subunit association or 16S RNA and 23S RNA association, some with both. These data clearly indicate that RNA-RNA interaction plays the major role in ribosomal subunit association.     

Binding of magnesium ions and ethidium bromide: comparison of ribosomes and free ribosomal RNA.

Comparative studies of free ribosomal RNA and ribosomes were made with two probes, Mg++ ions and ethidium bromide, which interact with RNA in different ways. Mg++. E. coli 16 S rRNA and 30 S ribosomes were equilibrated with four different buffers. Equilibration required several days at 4 degrees and several hours at 37 degrees. In all buffers ribosomes bound more Mg than free rRNA, the difference sometimes reaching 20--30%. Ribosomes were more resistant than free rRNA to heat denaturation and their denaturation was more highly cooperative. Ribosomes that bound more Mg++ had higher denaturation temperatures. Ethidium bromide. Fluorescence enhancement studies of ethidium intercalation showed the free 16 S rRNA to have 50--80 binding sites per molecule. A large fraction of these sites were present and accessible in the ribosome, but their ethidium-binding constants were reduced by an order of magnitude. In addition, free rRNA contained a small number of very strong binding sites that were virtually absent in the ribosomes.     

The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.     

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.     

Conformational change of 23S RNA in 50S ribosome is responsible for translocation in protein synthesis

Since the recognition of the ‘translocation’ phenomenon during protein synthesis several theories have been proposed, without much success, to explain the translocation of peptidyl tRNA from the aminoacyl site to the peptidyl site. The involvement of L7/L12 proteins and therefore the L7/L12 stalk region of 50S ribosomes in the translocation process has been widely accepted. The mobility of the stalk region, as recognised by many workers, must be of physiological significance. It has recently been shown in this laboratory that 50S ribosomes derived from tight and loose couple 70S ribosomes differ markedly in quite a few physical and biological properties and it appears that these differences are due to the different conformations of 23S RNAs. It has also been possible to interconvert tight and loose couple 50S ribosomes with the help of the agents, elongation factor -G, GTP (and its analogues) which are responsible for translocation. Thus loose couple 70S ribosomes so long thought to be inactive ribosomes are actually products of translocation. Further, the conformational change of 23S RNA appears to be responsible for the interconversion of tight and loose couple 50S ribosomes and thus the process of translocation. A model has been proposed for translocation on the basis of the direct experimental evidences obtained in this laboratory.     

Distances between 3' ends of ribosomal ribonucleic acids reassembled into Escherichia coli ribosomes

The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3' ends of the RNAs in 70S ribosomes were estimated by nonradiative energy transfer from fluorescein to eosin. The percentage of energy transfer was calculated from the decrease in fluorescence lifetime of fluorescein in the quenched sample compared to the unquenched sample. Fluorescence lifetime was measured in real time by using a mode-locked laser for excitation and a high-speed electrostatic photomultiplier tube for detection of fluorescence. The distances between fluorophores attached to the 3' ends of 16S RNA and 5S RNA or 23S RNA were estimated to be about 55 and 71 A, respectively. The corresponding distance between the 5S RNA and 23S RNA was too large to be measured reliably with the available probes but was estimated to be greater than 65 A. Comparison of the quantum yields of the labeled RNAs free in solution and reconstituted into ribosomal subunits suggests that the 3' end of 16S RNA does not interact appreciably with other ribosomal components and may be in a relatively exposed position, whereas the 3' ends of the 5S RNA and 23S RNA may be buried in the 70S ribosomal subunit.     

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.     

Protection of ribosomal RNA from kethoxal in polyribosomes: Implication of specific sites in ribosome function

In an attempt to probe the topography of 5 S, 16 S and 23 S RNAs in a functionally engaged ribosome, polysomes were probed using the structure-sensitive, guanine-specifie reagent kethoxal. Reactivities of guanine residues at 38 specific ribosomal RNA sites in polysomes were compared with their corresponding reactivities in vacant 70 S ribosomes. No polysome-specific protection was seen for 5 S RNA. In 16 S RNA, positions 530, 693 or 1079, 966, 1338 and 1517 showed protection in polysomes; all of these sites have highly conserved primary and secondary structures, and include several methylated nucleotides. In 23 S RNA, polysome protection is seen at positions 277, 1071, 1475 or 2112, 2116 and 2751. We attribute polysome-specific protection either to direct contact of transfer RNA and/or messenger RNA with the protected sites or to tRNA and/or mRNA-induced changes in ribosome conformation involving the protected sites.     

Intrinsic structural differences between "tight couples" and Kaltschmidt-Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the "classical" twice NH(4)Cl-washed ribosomes, also known as loose couples (LC), or to the "tight couples" preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.     

Area of 16S ribonucleic acid at or near the interface between 30S and 50S ribosomes of Escherichia coli.

To determine the region of 16S ribonucleic acid (RNA) at the interface between 30 and 50S ribosomes of Escherichia coli, 30 and 70S ribosomes were treated with T1 ribonuclease (RNase). The accessibility of 16S RNA in the 5' half of the molecule is the same in 30 and 70S ribosomes. The interaction with 50S ribosomes decreases the sensitivity to T1 RNase of an area in the middle of 16S RNA. A large area near the 3' end of 16S RNA is completely protected in 70S ribosomes. The RNA near the 3' end of the molecule and an area of RNA in the middle of the molecule appear to be at the interface between 30 and 50S ribosomes. One site in 16S RNA, 13 to 15 nucleotides from the 3' end, normally inaccessible to T1 RNase in 30S ribosomes, becomes accessible to T1 RNase in 70S ribosomes. This indicates a conformational change at the 3' end of 16S RNA when 30S ribosomes are associated with 50S ribosomes.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

Localization of the elongation factor Tu binding site on Escherichia coli ribosomes

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

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.     

Ribosomes and ribonucleic acids of Coxiella burneti

This report describes the direct isolation and characterization of rickettsial ribosomes. Ribosomes from the rickettsia Coxiella burneti were isolated and partially characterized. The ribosomes had a sedimentation constant of about 70S and could be dissociated into 50 and 30S subunits. Electron microscopy revealed ribosomal particles with dimensions similar to those reported for other procaryotic organisms. Ribonucleic acid (RNA) species (23 and 16S) were isolated from the ribosomal particles. The nucleotide compositions of the ribosomal RNAs were found to be similar to those reported for bacterial ribosomal RNA. In addition to the high-molecular-weight ribosomal RNA, 5S RNA was also extracted from the organism.