Stimulation of peptidyltransferase reactions by a soluble protein

The requirements for peptide-bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined using fMet-tRNA as donor substrate and puromycin or alpha-hydroxypuromycin as acceptors. We report that the soluble protein EF-P, purified to apparent homogeneity, stimulates the synthesis of N-formylmethionylpuromycin or N-formylmethionylhydroxypuromycin by 70S ribosomes or reassociated 30S and 50S subunits. In the presence of EF-P, 70S ribosomes are significantly more efficient than 50S particles in catalysing either peptide-bond synthesis or transesterification. The involvement of 50S subunit proteins in EF-P-stimulated peptide-bond formation and transesterification was studied. 50S subunits were dissociated by 2.0 M LiCl into core particles and 'split' proteins, several of which were purified to homogeneity. When added to 30S X A-U-G X f[35S]Met-tRNA, 50S cores or 50S cores reconstituted with L6 or L11 promoted peptide-bond synthesis or transesterification poorly. EF-P stimulated peptide-bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF-P stimulated a low level of transesterification by cores reconstituted with L6 and L11. In contrast, core particles reconstituted with L16 exhibited both peptide-bond-forming and transesterification activities and EF-P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF-P-stimulated peptide-bond and transesterification reactions of the peptidyl transferase. Ethoxyformylation of either 50S subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide-bond formation and transesterification. Similarly ethoxyformylation of EF-P resulted in a 25-50% loss of its ability to stimulate both reactions. 30S subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF-P in the catalytic mechanism of peptidyltransferase is discussed.     

The Escherichia coli ribosomal protein L11 suppresses release factor 2 but promotes the release factor 1 activities in peptide chain termination

The incubation of the 50 S ribosomal subunits of Escherichia coli with 1.5 M LiCl yields 1.5c core particles depleted in 14 proteins and inactive in peptide chain termination. In codon-dependent peptidyl-tRNA hydrolysis the release factor 1 (RF-1)-induced reaction essentially depends on both L11 and L16 whereas the release factor 2 (RF-2)-induced reaction is depressed by L11 and stimulated by L16. Omission of L11 results in a several-fold increase in the specific activity of the RF-2. Functional complexes are formed with RF-2 at an apparent Km (dissociation constant) for the termination codon 5-fold lower than with reconstituted ribosomes containing L11; the Vmax for the hydrolysis is unchanged. L11 suppresses this effect when added to the core at close to molar equivalence. In contrast, RF-1 has a very low activity if ribosomes lack L11 and this can be restored by titration of L11 back to the core. This is the first example of a differential or an opposite effect of a ribosomal component on the activities of the two release factors, and the studies suggest that L11 has a critical role in the binding domain for the two factors.     

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.     

L16, a bifunctional ribosomal protein and the enhancing effect of L6 and L11

L16 exhibits both peptide bond and transesterification activities when reconstituted into 2 M LiCl core particles. L6 and L11, when reconstituted in a similar manner in the absence of L16, manifest significant transesterification activity. Both L6 and L11 enhance the transesterification activity of L16; L11 being more active than L6 in this respect. However, both L6 and L11 have minimal effect on peptide bond formation when reconstituted with L16 at concentrations more than 2.5 M equivalents. Both L6 and L11 exhibit a differential effect on transesterification. The affinity-labelling agents, like PhCH2SO2F, diisopropylfluorophosphate and ethoxyformic anhydride, have been used to explore the role of residues in peptide bond formation and transesterification. It is proposed that the Ser-Phe combination present in L16, L11 and L6 is involved in transesterification in addition to the single histidine in L16. The single histidine in L16 appears to be important in the catalysis of peptide bond formation and transesterification.     

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.     

The importance of the Escherichia coli ribosomal protein L16 for the reconstitution of the peptidyl-tRNA hydrolysis activity of peptide chain termination

The incubation of the 50 S ribosomal subunit of Escherichia coli with 1.5 M LiCl yields 1.5c core particles inactive in the peptidyl-tRNA hydrolysis activity of in vitro termination. The omission of L16 alone from reconstitutions of the proteins into the core results in inactive ribosomes. The single omission of a number of other proteins, in particular L7/L12, L10, L25, L27, and L15, gives ribosomes with intermediate activity. L16 alone is unable to restore significant activity to 1.5c cores, but together L16 and the above "stimulating" proteins produce particles as active as those reconstituted with the full complement of proteins. The ribosomal proteins important for the expression of peptidyl-tRNA hydrolysis and peptidyl transferase activities are very similar. However, ribosomes lacking both L11 and L16, but not L16 alone, surprisingly can catalyze codon- and release factor 2-dependent peptidyl-tRNA hydrolysis. The addition of L16 dramatically increases the activity. L16 is, therefore, important but not essential for the expression of the release factor 2-dependent peptidyl-tRNA hydrolysis.     

The peptidyltransferase centre of the Escherichia coli ribosome. The histidine of protein L16 affects the reconstitution and control of the active centre but is not essential for release-factor-mediated peptidyl-tRNA hydrolysis and peptide bond formation

Modification of the Escherichia coli 50S ribosomal subunit with histidine-specific diethyl pyrocarbonate affects peptide bond formation and release-factor-dependent peptidyl-tRNA hydrolysis. Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre. When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16. The modified protein does mediate the partial restoration of peptide bond formation and release-factor-2 activities to these particles. It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle. A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl-tRNA hydrolysis. L16 stimulates these activities. A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16. The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.     

Reversible modification of Escherichia coli ribosomes with 2,3-dimethylmaleic anhydride. A new method to obtain protein-deficient ribosomal particles.

Treatment of Escherichia coli ribosomes with the protein reagent 2,3-dimethylmaleic anhydride is accompanied by inactivation of polypeptide polymerization and by dissociation of ribosomal proteins. Regeneration of the modified amino groups at pH 6.0 is followed by reactivation and reconstitution of the ribosomes. Prior to regeneration of the amino groups, ribosomal particles and split proteins can be separated by centrifugation, which allows the preparation of new protein-deficient particles. The ribosomal particles obtained by three successive treatments with 2,3-dimethyl-maleic anhydride at a molar ratio of reagent to ribosome equal to 16,000 lack proteins S1, S2, S3, S5, S10, S13, S14, L7, L8, L10, L11, L12, and L20 and have lost part of proteins S4, L1, L6, L16, and L25. This new procedure to obtain protein-deficient ribosomal particles is mild and might be useful to dissociate other protein-containing structures in addition to 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.     

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.     

Proteins fro Escherichia coli ribosomes involved in the binding of erythromycin.

When 50 S subunits from Escherichia coli ribosomes were incubated with 1·3 m-LiC1 the resulting 1·3c core was inactive both with respect to peptidyltransferase activity and erythromycin binding (tested by equilibrium dialysis). Reconstitution experiments with purified proteins from the corresponding split fraction SP1·3 revealed that only L16 (reconstituted with the 1·3c core in a tenfold excess) could restore high activity in both systems.When 30 out of the 34 isolated ribosomal proteins were tested directly for binding or erythromycin, L15 was able to bind the drug, in contrast to all other proteins including L16. Total reconstitution experiments with the 50 S subunit demonstrated an absolute requirement for L15 and L16 with respect to both drug binding and peptidyltransferase activity.     

Reconstitution of the active rat liver 60 S ribosomal subunit from different preparations of core particles and split proteins

Proteins extracted from the 60 S rat liver ribosomal subunit with 50% ethanol/0.5 M K Cl produced only a partial reactivation of the corresponding core particles. In contrast, the same split proteins were able to reactivate the core particles prepared with dimethyl-maleic anhydride (DMMA) to the same level as that observed using the DMMA-split proteins, i.e. 60-80% of the control according to the catalytic activities tested. Comparative analysis of the two split protein fractions showed only four common proteins: P1-P2, which alone restored part of the activities, especially the EF-2-dependent GTPase one, and L10a, L12, which must be responsible for the additional reactivation. The poor ability of the ethanol/KCl core particles to be reactivated was shown to be probably related to a conformational alteration which destabilized the 5 S RNA-protein complex. Proteins present in the ethanol/KCl wash of Saccharomyces cerevisiae 60 S subunits were found to be partly active in subunit reconstitution using rat liver DMMA core particles.     

The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.     

Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits.

An ethionine-containing submethylated particle related to the 50 S ribosomal subunit has been isolated from Escherichia coli grown in the presence of ethionine. This particle (E-50S) lacks L16, contains reduced amounts of L6, L27, L28 and L30 and possesses a more labile and flexible structure than the normal 50 S subunit. The E-50S particle has defective association properties and is incapable of peptide bond formation. It can be converted to an active 50 S ribosomal subunit when ethionine-treated bacteria are incubated under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin).Total reconstitution of 50 S ribosomal subunits in vitro using normal 23 S and 5 S ribosomal RNA and proteins prepared from E-50S particles yields active subunits only if L16 is also added. The hypothesis that E-50S particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a late stage (L16 integration) in the normal 50 S assembly process is discussed.     

An efficient in vitro total reconstitution of the Escherichia coli 50S ribosomal subunit

A new, relatively simple technique for the total invitro reconstitution of E. coli 50S ribosomes has been developed. It is a two-step procedure like that previously reported by Nierhaus and Dohme [Proc. Natl. Acad. Sci. 71, 4713 (1974)], but it differs in a number of important aspects. Ribosomal RNA is prepared by direct phenol extraction of 70S particles to minimize nuclease fragmentation. A mixture of 50S proteins is prepared by acetic acid extraction and immediate removal of the acetic acid by thin film dialysis. The resulting protein mixture is soluble and stable. Separate RNA and protein fractions are mixed, incubated first at 44 degrees C in 7.5 mM Mg(2+), and then at 50 degrees C in 20 mM Mg(2+). The resulting 50S particles comigrate with native 50S particles in analytical gradients. They range from 50 to 100% active in five different functional assays. This is a fairly stringent test of the effectiveness of reconstitution since 50S particles derived from highly active vacant couples were used as a control.Images     

30S ribosomal subunits with fragmented 16S RNA: a new approach for structure and function study of ribosomes.

A new approach for function and structure study of ribosomes based on oligodeoxyribonucleotide-directed cleavage of rRNA with RNase H and subsequent reconstitution of ribosomal subunits from fragmented RNA has been developed. The E coli 16S rRNA was cleaved at 9 regions belonging to different RNA domains. The deletion of 2 large regions was also produced by cleaving 16S rRNA in the presence of 2 or 3 oligonucleotides complementary to different RNA sites. Fragmented and deleted RNA were shown to be efficiently assembled with total ribosomal protein into 30S-like particles. The capacity to form 70S ribosomes and translate both synthetic and natural mRNA of 30S subunits reconstituted from intact and fragmented 16S mRNA was compared. All 30S subunits assembled with fragmented 16S rRNA revealed very different activity: the fragmentation of RNA at the 781-800 and 1392-1408 regions led to the complete inactivation of ribosomes, whereas the RNA fragmentation at the regions 296-305, 913-925, 990-998, 1043-1049, 1207-1215, 1499-1506, 1530-1539 did not significantly influence the ribosome protein synthesis activity, although it was also reduced. These findings are mainly in accordance with the data on the functional activity of some 16S rRNA sites obtained by other methods. The relations between different 16S RNA functional sites are discussed.     

Multiple defects in translation associated with altered ribosomal protein L4

The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribosomes carrying the altered L4 protein support increased levels of frameshifting, missense decoding and readthrough of stop codons during the elongation phase of protein synthesis and stimulate utilization of non-AUG codons and mutant initiator tRNAs at initiation. L4 mutant ribosomes are also altered in their interactions with a range of 30S-targeted antibiotics. In contrast, the L22 mutant is relatively unaffected in both decoding activities and antibiotic interactions. These results suggest that mutations in the large subunit protein L4 not only alter the structure of the 50S subunit, but upon subunit association, also affect the structure and function of the 30S subunit.     

Effects of aminoethylation of cysteine residues on the structural and functional activities of the Escherichia coli 30 S ribosomal proteins

The purified 30 S ribosomal proteins from Escherichia coli strain Q13 were chemically modified by reaction with ethyleneimine, specifically converting cysteine residues to S-2-aminoethylcysteine residues. Proteins S1, S2, S4, S8, S11, S12, S13, S14, S17, S18 and S21 were found to contain aminoethylcysteine residues after modification, whereas proteins S3, S5, S6, S7, S9, S10, S15, S16, S19 and S20 did not. Aminoethylated proteins S4, S13, S17 and S18 were active in the reconstitution of 30 S ribosomes and did not have altered functional activities in poly(U)-dependent polyphenylalanine synthesis, R17-dependent protein synthesis, fMet-tRNA binding and Phe-tRNA binding. Aminoethylated proteins S2, S11, S12, S14 and S21 were not active in the reconstitution of complete 30 S ribosomes, either because the aminoethylated protein did not bind stably to the ribosome (S2, S11, S12 and S21) or because the aminoethylated protein did not stabilize the binding of other ribosomal proteins (S14). The functional activities of 30 S ribosomes reconstituted from a mixture of proteins containing one sensitive aminoethylated protein (S2, S11, S12, S14 or S21) were similar to ribosomes reconstituted from mixtures lacking that protein. These results imply that the sulfhydryl groups of the proteins S4, S13, S17 and S18 are not necessary for the structural or functional activities of these proteins, and that aminoethylation of the sulfhydryl groups of S2, S11, S12, S14 and S21 forms either a kinetic or thermodynamic barrier to the assembly of active 30 S ribosomes in vitro.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.