Yeast ribosomal protein deletion mutants possess altered peptidyltransferase activity and different sensitivity to cycloheximide.

The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal peptidyltransferase. Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit. We have developed an in vitro system for the determination of peptidyltransferase activity in yeast ribosomes. Using this system, a kinetic analysis of a model reaction for peptidyltransferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromycin. This reaction (puromycin reaction) followed first-order kinetics. At saturating concentrations of puromycin, the first-order rate constant (k(3)) is identical to the catalytic rate constant (k(cat)) of peptidyltransferase. This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 degrees C. We now present for the first time kinetic evidence that yeast ribosomes lacking a particular protein of the 60S subunit may possess significantly altered peptide bond-forming ability. The k(cat) of peptidyltransferase from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 min(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1). These results suggest that the presence of ribosomal proteins L24 and, to a lesser extent, L39 is required for exhibition of the normal catalytic activity of the ribosome. Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis. However, the absence of L24 caused increased resistance to cycloheximide, a translocation inhibitor. Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 microM cycloheximide for the L24 mutant compared to 0.7 microM for the wild type.     

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling

The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.     

Biochemical and kinetic characteristics of the interaction of the antitumor antibiotic sparsomycin with prokaryotic and eukaryotic ribosomes

Using 125I-labeled phenol-alanine sparsomycin, an analogue of sparsomycin having higher biological activity than the unmodified antibiotic, we studied the requirements and the characteristics of its interaction with the ribosome. The drug does not bind to either isolated ribosomal subunits or reconstituted whole ribosomes. For sparsomycin binding to 70S and 80S ribosomes, the occupation of the peptidyltransferase P-site by an N-blocked aminoacyl-tRNA is a definitive requirement. The sparsomycin analogue binds to bacterial and yeast ribosomes with Ka values of around 10(6) M-1 and 0.6 x 10(6) M-1, respectively, but its affinity is probably affected by the character of the peptidyl-tRNA bound to the P-site. Chloramphenicol, lincomycin, and 16-atom ring macrolides compete with sparsomycin for binding to bacterial ribosomes, but streptogramins and 14-atom ring macrolides do not. Considering the reported low affinity of puromycin for bacterial ribosomes, this antibiotic is also a surprisingly good competitor of sparsomycin binding to these particles. In the case of yeast ribosomes, blasticidin is a relatively good competitor of sparsomycin interaction, but anisomycin, trichodermin, and narciclasin are not. As expected, puromycin is a poor competitor of the binding in this case. The results from competition studies carried out with different sparsomycin analogues reveal, in some cases, a discrepancy between the drug ribosomal affinity and its biological effects. This suggests that some intermediate step, perhaps a ribosomal conformational change, is required for the inhibition to take place.     

Effects of two photoreactive spermine analogues on peptide bond formation and their application for labeling proteins in Escherichia coli functional ribosomal complexes.

The effect of two photoreactive analogues of spermine, N(1)-azidobenzamidino- (ABA-) spermine and N(1)-azidonitrobenzoyl- (ANB-) spermine, on ribosomal functions was studied in a cell-free system derived from Escherichia coli. In the dark, both analogues stimulated the binding of AcPhe-tRNA to poly(U)-programmed ribosomes, enhanced the stability of the ternary complex AcPhe-tRNA.poly(U).ribosome (complex C), and caused stimulatory and inhibitory effects on peptidyltransferase activity. ABA-spermine exhibited more pronounced effects than ANB-spermine. Each photoprobe was covalently attached after irradiation to both ribosomal subunits and also to free rRNA isolated from 70S ribosomes. Photolabeled complex C showed a reactivity toward puromycin, similar to that exhibited by complex C reacting reversibly with photoprobes free in solution. The distribution of the incorporated radioactivity among the ribosomal components was determined under two experimental conditions, one stimulating and the other inhibiting peptidyltransferase activity. Under both conditions, ABA-spermine was the strongest cross-linker. Upon stimulatory conditions, 14% of ABA-[(14)C]spermine cross-linked to complex C was bound to the protein fraction. The proteins primarily labeled were identified as S3, S4, L2, L3, L6, L15, L17, and L18. Upon inhibitory conditions, a higher percent of the incorporated radioactivity was found in ribosomal proteins, while the pattern of protein labeling was characterized by a remarkable decrease of cross-linked proteins L2, L3, L6, L15, L17. and L18 and by an increase of cross-linked proteins S9, S18, L1, L16, L22, L23, and L27. On the basis of these results and literature data, the involvement of spermine in the conformation and important functions of ribosomes is discussed.     

The mechanism of covalent reaction of bromoacetyl-phenylalanyl-transfer RNA with the peptidyl-transfer RNA binding site of the Escherichia coli ribosome

Results are presented to prove that bromoacetyl-phenylalanyl-transfer RNA reacts covalently with 50 S ribosomal proteins L2 and L27 while it is bound correctly to the peptidyl site on the 70 S ribosome. Attachment of the BrAcPhe moiety to tRNA causes a 100-fold enhancement of its reactivity with ribosomes. This reactivity closely parallels binding of tRNA whether measured by poly(U) stimulation or competition with deacylated tRNA. BrAcPhe-tRNA can bind correctly to the P site as judged by puromycin releasibility and lack of tetracycline inhibition. Little significant reaction of BrAcPhe-tRNA with L2 and L27 occurs during procedures used to purify and analyze ribosomal proteins. If ribosomes are first incubated with BrAcPhe-tRNA and subsequently treated with puromycin before analysis, little inhibition of the covalent reaction with L2 and L27 is observed. In contrast, a few minor reaction products are markedly suppressed. Covalently attached BrAcPhe-tRNA is still capable of accepting an amino acid from Phe-tRNA or puromycin. The products from this reaction are found attached to proteins L2 and L27 and to a lesser extent to L15 and L16. This shows that true affinity labeling of proteins in the peptidyl binding site has been accomplished.Some covalent reaction of BrAcPhe-tRNA with the 30 S protein S18 is also observed. This reaction is not poly(U)-dependent, however, and S18-reacted BrAcPhe-tRNA is not capable of peptide bond formation with Phe-tRNA. It seems likely that reaction with S18 results from a non-functional interaction of the affinity label with the ribosome.     

Affinity labeling of the P site of Drosophila ribosomes: a comparison of results from (bromoacetyl)phenylalanyl-tRNA and mercurated fragment affinity reactions

The binding site of the peptidyl group of peptidyl-tRNA in the P site of Drosophila ribosomes was probed with (bromoacetyl)phenylalanyl-tRNA (BrAcPhe-tRNA). This affinity label binds specifically to the P site by virtue of its ability to participate in peptide bond formation with puromycin following its attachment to ribosomes. As many as nine ribosomal proteins may be labeled under these conditions; however, the majority of the labeling is associated with three large-subunit proteins and two small-subunit proteins. Two of the large-subunit proteins, L4 and L27, are electrophoretically very similar to the proteins labeled by the same reagent in Escherichia coli ribosomes L2 and L27. Reexamination by a different two-dimensional gel system of the ribosomal components labeled by a second P site reagent, the 3' pentanucleotide fragment of N-acetylleucyl-tRNA which is derivatized to contain mercury atoms at the C-5 position of all three cytosine residues, shows two major and three minor labeled proteins. These proteins, L10/L11, L26, S1/S4, S13, and S20, are likely present in the binding site of the 3' end of peptidyl-tRNA, a site that appears to span both subunits. These results have allowed us to construct a model for the protein positions in and near the peptidyl-tRNA binding site of Drosophila ribosomes.     

Identification of proteins at the peptidyl-tRNA binding site of rat liver ribosomes

Summary We have identified proteins involved in the peptidyl-tRNA-binding site of rat liver ribosomes, using an affinity label designed specifically to probe the P-site in eukaryotic peptidyl transferase. The label is a 3-terminal pentanucleotide fragment of N-acetylleucyl-tRNA in which mercury atoms have been added at the C-5 position of the three cytosine residues. This mercurated fragment can bind to rat liver peptidyl transferase and function as a donor of N-acetylleucine to puromycin. Concommitant with this binding, the mercury atoms present in the fragment can form a covalent linkage with a small number of ribosomal proteins. The major proteins labeled by this reagent are L5 and L36A. Four protein spots are found labeled to a lesser extent: L10, L7/7a, L3/4 and L25/31. Each of these proteins, therefore, is implicated in the binding of the 3-terminus of peptidyl-tRNA.The results presented here are correlated with other investigations of the structure-function aspects of rat liver peptidyl transferase. Using these data, we have constructed a model for the arrangement of proteins within this active site.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.     

Studies on the catalytic rate constant of ribosomal peptidyltransferase

A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin. This reaction (puromycin reaction) is first order at all concentrations of S tested. An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and [S] shows hyperbolic saturation and that the value of kobs at saturating [S] is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction. With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence. The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence. This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro. The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex.     

Active center cleft residues of pokeweed antiviral protein mediate its high-affinity binding to the ribosomal protein L3

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP) which catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA and thereby inhibits the protein synthesis. The ribosomal protein L3, a highly conserved protein located at the peptidyltransferase center of the ribosomes, is involved in binding of PAP to ribosomes and subsequent depurination of the SRL. We have recently discovered that recombinant PAP mutants with alanine substitution of the active center cleft residues (69)NN(70) (FLP-4) and (90)FND(92) (FLP-7) that are not directly involved in the catalytic depurination at the active site exhibit >150-fold reduced ribosome inhibitory activity [(2000) J. Biol. Chem. 275, 3382--3390]. We hypothesized that the partially exposed half of the active site cleft could be the potential docking site for the L3 molecule. Our modeling studies presented herein indicated that PAP residues 90--96, 69--70, and 118--120 potentially interact with L3. Therefore, mutations of these residues were predicted to result in destabilization of interactions with rRNA and lead to a lower binding affinity with L3. In the present structure-function relationship study, coimmunoprecipitation assays with an in vitro synthesized yeast ribosomal protein L3 suggested that these mutant PAP proteins poorly interact with L3. The binding affinities of the mutant PAP proteins for ribosomes and recombinant L3 protein were calculated from rate constants and analysis of binding using surface plasmon resonance biosensor technology. Here, we show that, compared to wild-type PAP, FLP-4/(69)AA(70) and FLP-7/(90)AAA(92) exhibit significantly impaired affinity for ribosomes and L3 protein, which may account for their inability to efficiently inactivate ribosomes. By comparison, recombinant PAP mutants with alanine substitutions of residues (28)KD(29) and (111)SR(112) that are distant from the active center cleft showed normal binding affinity to ribosomes and L3 protein. The single amino acid mutants of PAP with alanine substitution of the active center cleft residues N69 (FLP-20), F90 (FLP-21), N91 (FLP-22), or D92 (FLP-23) also showed reduced ribosome binding as well as reduced L3 binding, further confirming the importance of the active center cleft for the PAP--ribosome and PAP--L3 interactions. The experimental findings presented in this report provide unprecedented evidence that the active center cleft of PAP is important for its in vitro binding to ribosomes via the L3 protein.     

Modifications of ribosomes from rat liver with alkylating derivatives of tRNA]

Alkylating analogs of peptidyl-tRNA: N-chloroambucilyl-14C-phenylanalyl-tRNA (1), N-iodoacetyl-14C-phenylalanyl-tRNA (2) and N-bromo-acetyl-14C-phenlalanyl-tRNA (3) were applied for the modification of the peptidyl-transferase center of the 80S ribosomes from rat liver. These analogs, being in the teronary complex poly-U: ribosome : tRNA analog, modified ribosomal proteins and ribosomal RNA. The modification is directed to large ribosomal subunit. It is found, that (1) modifies ribosomal proteins L5, L25, L31 and L32 and (2) modifies ribosomal proteins L4, L6, L10+L11, L13 and L30.     

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.     

Sequence of the amino-terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a,L18, L27, L30, L37, L37a,and L39

The sequence of the amino-terminal region of eleven rat liver ribosomal proteins–S4, S6, S8, L7a, L18, L27, L30, L37a, and L39 - was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.     

Affinity labelling of rat liver ribosomal protein S26 by heptauridylate containing a 5′-terminal alkylating group

Heptauridylate bearing a radioactive alkylating [¹⁴C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to the 5-phosphate via amide bond, was bound to ribosomes and small ribosomal subunits from rat liver which thereby were coded to bind N-acylated Phe tRNA. After completion of the alkylating reaction and subsequent hydrolysis of the phosphamide bond ribosomal proteins were isolated. Radioactivity was found covalently associated preferentially with protein S26 and, to a very small extent, with proteins S3 and S3a. The affinity labelling reaction could be abolished by (pU)₁₄ and poly(U). From the results it is concluded that ribosomal protein S26 is located at the mRNA binding site of rat liver ribosomes.     

Affinity labelling of yeast ribosomal peptidyl transferase

Summary Using p-nitrophenylcarbamyl-phenylananyl-tRNA (PNPC-Phe-tRNA) and N-Iodoacetyl-phenylalanyl-tRNA as affinity labels we have attempted to identify the components of the aminoacyl-tRNA binding sites located in the vicinity of the peptidyl transferase centre of the yeast ribosome. Both Phe-tRNA derivatives bind to the ribosomal A-site in the presence of 20 mM Mg⁺⁺ ion concentration and can be translocated to the ribosomal P-site in the presence of elongation factor. After the labels have been allowed to react covalently with ribosomes they were found associated with the large ribosomal subunit. Proteins L36, L43, L42, L29, L2, L17/18, L19/20 and proteins L26, L38, L22/23, L7/9, L4/6, L36, L11, L43, L39 were labelled in samples treated with PNPC-Phe-tRNA and N-Iodoacetyl-Phe-tRNA respectively. In contrast, when only the components of the ribosomal P-site were analysed by reacting the treated particles with puromycin fewer spots were labelled, corresponding to proteins L36 and L19/20 using PNPC-Phe-tRNA and proteins L4/6, L36, and L43 using N-Iodoacetyl-Phe-tRNA.     

Comparison of the effect of various methods of dissociation on the protein composition of rat liver ribosomal subunits.

Rat liver ribosomes were dissociated into subunits using EDTA, sodium pyrophosphate, high concentrations of KC1, as well as by incubation with puromycin in presence of 0.5 M KC1. The subunits obtained were analyzed using the density gradient centrifugation technique and their ribosomal proteins were separated by means of two-dimensional polyacrylamide gel electrophoresis. The ribosomal protein patterns of the two subunits isolated using each of the dissociating method were compared to the protein patterns of monosomes prepared by puromycin treatment alone. Our results revealed that the use of chelating agents to dissociate the ribosomes resulted in the loss of some ribosomal proteins from the small subunit. On the other hand, the use of KC1 in high concentrations to dissociate the ribosomes did not appear to cause any major loss of proteins from the ribosomes except for some acidic proteins.     

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.     

Oxazolidinones mechanism of action: inhibition of the first peptide bond formation

Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steady-state kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA(i)(Met) to the ribosomal peptidyltransferase P-site, which is vacant only prior to the formation of the first peptide bond.     

Ribosome conformational changes associated with protein S6 phosphorylation

The relative accessibility of rat liver ribosomal proteins to reductive methylation was examined using ribosomes with unphosphorylated, and extensively phosphorylated S6. Comparison of the results indicated that proteins S3, S4, S7, and S23/24 of the small subunit, and proteins L9, L10, L12, L18, L27, L34, and L36 are involved in a ribosomal conformational change.