Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.     

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.     

Ribosomal activity of the 16 S.23 S RNA complex

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

Biosynthesis of vitamin B-12 Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K₁₂ and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[⁵⁸Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.     

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.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.     

Specific release of ribosomal proteins by nucleic acid-intercalating agents.

Increasing concentrations of ethidium bromide cause progressive inactivation of ribosomes, apparently by binding to double-stranded regions of the rRNA. At low drug concentrations (10(-4)M) the partial inhibition detected is due to specific release of proteins L7 and L12; activity can be restored by addition of an excess of these two proteins. At higher concentrations the inactivation is not reversed by supplementation with released proteins. The presence of ethanol affects the extent of ethidium binding and also the release of ribosomal proteins. In all tests the proteins most sensitive to the presence of the drug are L7 and L12, followed by L8/9, L11, L27, L28, L29 and L30. Despite the fact that L7 and L12 are the first two proteins released by ethidium they are never totally missing from drug-treated ribosomes, though the other proteins can be displaced completely. About 50% of proteins L7 and L12 remain on the ribosomes at the highest drug concentrations tested, possibly indicating heterogeneity in the binding sites for the several copies present in the ribosome.     

50-S subunit from Escherichia coli ribosomes. Isolation of active ribosomal proteins and protein complexes.

A method is described for the isolation of highly purified proteins from the 50-S subunit of Escherichia coli ribosomes. All the proteins from the large subunit could be isolated with the exception of L14, L26, L31 and L34. The isolated proteins are functionally active in reconstituted particles. The method consists of successive NH4Cl/EtOH and LiCl washing steps, which split off distinct groups of proteins from the ribosome. The protein groups are further separated by a combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (carboxymethylcellulose) in the presence of 6 M urea, at neutral pH and 4 degrees C. The purity of the proteins was analyzed by two-dimensional gel electrophoresis. In addition, ten protein complexes were isolated and identified.     

Dissociation of intact Escherichia coli ribosomes in a mass spectrometer. Evidence for conformational change in a ribosome elongation factor G complex

We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding. From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10). Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA. Replacement of Mg(2+) by Li(+) ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins. Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation. By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate. By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA. These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions.     

Phosphorylation in vitro and in vivo of ribosomal proteins from Saccharomyces cerevisia.

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [gamma-32P]ATP. Analysis of the ribosomal proteins with two-dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 M KCl and was not dependent on adenosine 3':5'-monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones. Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high-voltage electrophoresis. After two-dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.     

Mechanism and site of action of a ribosome-inactivating protein type 1 from Dianthus barbatus which inactivates Escherichia coli ribosomes.

A single chain ribosome-inactivating protein with RNA N-glycosidase activity, here named Dianthin 29, was isolated from leaves of Dianthus barbatus L. Incubation of intact Escherichia coli ribosomes with Dianthin 29 and subsequent aniline treatment of the isolated rRNA releases a rRNA fragment of 243 nucleotides from 23 S rRNA. Nucleotide sequence studies showed that the site of N-glycosidic bond cleavage is at A-2660 within the universally conserved sequence 5'-AGUACGAGAGGA-3' near the 3'-end of 23/28 S rRNAs. To our knowledge, Dianthin 29 is the first ribosome-inactivating protein which is shown to inactivate intact prokaryotic ribosomes in the same manner as eukaryotic ribosomes.     

Acidic ribosomal proteins from eukaryotic cells. Effect on ribosomal functions.

Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be substituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.     

Localisation of part of the binding sites of 30S ribosomal proteins S4 and S20 in a small uninterrupted fragment of 16S RNA.

Analyses of the T1 ribonuclease-alkaline phosphatase fingerprint of a continuous fragment of the 16S rRNA, 170-230 nucleotides long, isolated from the products of autodigestion of 30S ribosome subunits show that it contains a sequence near the 5'-phosphate terminus of intact 16S rRNA and corresponds to segment H'-M of this molecule as defined by Ehresmann et al [29]. Incubation of this fragment with total 30S ribosomal proteins under reconstitution conditions leads to the formation of a complex containing proteins S4, S20, and one or both of proteins S16 and S17. The stoichiometry of these proteins in the complex is discussed.     

Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding.

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.     

Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli.

The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.     

Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.     

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.