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.     

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.     

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.     

The effect of magnesium starvation on the dissociation of ribosomal proteins from Escherichia coli K-12 ribosomes.

The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli. During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit. An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus [5] as tightly bound. Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+. These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits.     

Further studies on the identity of proteins at the subunit interface of the 70 S E. coli ribosome

The ability of 1 m NH₄Cl to detach iodinated 50 S ribosomal proteins from 50 S subunits and 70 S ribosomes was compared. High salt treatment was effective in preferentially releasing L16, L20, L24, L26, L27, L29, and L30 from the 50 S subunit. Similar but smaller effects were seen for L2, L6, L15, L19, L28, and L31. When these results are combined with several previous studies on accessibility, twelve 50 S proteins appear to be less exposed in the 70 S particle than in the free subunit, by more than one entirely different measure of accessibility. These twelve must be considered strong candidates for possible subunit interface proteins.Lactoperoxidase catalyzed iodination was used to probe the surface topography of active and reversibly inactivated 30 S subunits. The magnesium depleted inactive 30 S particle reproducibly incorporates more ¹²⁵I than the active subunit indicating that a conformational change, characterized by an opening or expansion of the 30 S particles, accompanies 30 S inactivation. Seven 30 S proteins, S5, S21, S4, S7, S10, S13, and S16 become more accessible to lactoperoxidase as a result of inactivation. These proteins are different from those known to become more accessible to lactoperoxidase as a result of the conformational reorganization accompanying subunit association, S3, S6, S9, and S18. Thus, although both inactive 30 S and 50 S-bound 30 S are more open or reactive compared with free active 30 S, the regions which are affected appear to be different.     

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 50s ribosomal subunits or 70s ribosomes

Further to our studies on the Escherichia coli 30S ribosomal subunit, the detailed surface topography of both 50S subunits and 70S ribosomes has been investigated by using iodination catalyzed by immobilized lactoperoxidase as the surface probe. In the 50S subunit, only proteins L2, L5, L10, and L11 were iodinated to a significant and reproducible extent. The targets of iodination were identified, after isolation of the individual iodinated proteins, and were as follows: in protein L2 (271 amino acids), tyrosine-102 and -160; in protein L5 (178 amino acids), tyrosine-142; in protein L10 (165 amino acids), tyrosine-132; in protein L11 (142 amino acids), tyrosine-7 and -61. In the 70S ribosome, only protein L5 was still iodinated to a significant extent from the 50S subunit, whereas in the 30S subunit the same spectrum of iodinated proteins was observed as that from iodinated isolated 30S subunits, with the exception that S21 was no longer present.     

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.     

Subunit association defects in Escherichia coli ribosome mutants lacking proteins S20 and L11

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

Effect of polyamines on in vitro reconstitution of ribosomal subunits

The effect of polyamines on in vitro reconstitution of Escherichia coli 30S and 50S ribosomal subunits has been studied. Spermidine stimulated the reconstitution of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits at least 1.6-fold. The reconstitution of 30S particles from normal 16S rRNA and total proteins of 30S subunits exhibited only slight spermidine stimulation. However, the optimal Mg2+ concentration of the reconstitution was decreased from 20 mM to 16 mM in the presence of 3 mM spermidine. In the absence of spermidine the assembly of 30S particles from normal 16S rRNA was more rapid than the assembly from 16S rRNA lacking the methyl groups on two neighboring adenines. The reconstitution of 50S particles from 23S and 5S rRNA and total proteins of 50S subunits was not influenced greatly by spermidine. Gel electrophoresis results, from reconstitution experiments of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits, showed that the assembly of S1 and S9 proteins to 23S core particles was stimulated by spermidine during reconstitution. The relationship of polyamine effects on in vitro ribosome assembly from its constituents to in vivo ribosome assembly is discussed. The reconstitution of Bacillus subtilis 30S particles from 16S rRNA and total proteins of 30S subunits was also stimulated approximately 1.3-fold by 3 mM spermidine.     

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.     

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.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

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.     

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.     

Characterization of spinach plastid ribosomal proteins by two dimensional gel electrophoresis

Summary Ribosomes are isolated from spinach plastids using conventional sucrose gradients. Their subunits are prepared by dissociation using low Mg²⁺ concentration.It is shown that plastid ribosomes are able to bind f-met-tRNA in the presence of the initiation factors from E. coli.The characterization of ribosomal proteins is carried out using the four two-dimensional gel electrophoretic systems of Madjar et al. (1979). The 30 S and 50 S subunits contain 24 and 34 ribosomal poteins, respectively. These proteins are found in the 70S monosomes which also contain most often nine additional faintly stained 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.     

Activity of the 30-S CsCl core in elongation-factor-dependent GTP hydrolysis.

The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent FTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T-dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent (GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G-GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits.     

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.     

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cytosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two-to four-fold lower than that of cytoplasmic ribosomes.