Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit

The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtA(C) protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtA(C) does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtA(C) to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtA(C). Assays of epitope-tagged wild-type and mutant variants of CgtA(C) indicate that the C terminus of CgtA(C) is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtA(C) alleles to function in vivo. Depletion of CgtA(C) led to perturbations in the polysome profile, raising the possibility that CgtA(C) is involved in ribosome assembly or stability.     

Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors

Alpha-sarcin cleaves one phosphodiester bond of 23S rRNA within 70S ribosomes or 50S subunits derived from E. coli. The resulting fragment was isolated and sequenced. The cleavage site was identified as being after G2661 and is located within a universally conserved dodecamer. Cleavage after G2661 specifically blocked the binding of both elongation factors, i.e. that of the ternary complex Phe-tRNA*EF-Tu*GMPPNP and of EF-G*GMPPNP, whereas all elongation-factor independent functions of the ribosome, such as association of the ribosomal subunits, tRNA binding to A and P sites, the accuracy of tRNA selection at both sites, the peptidyl transferase activity, and the EF-G independent, spontaneous translocation, were not affected at all. Control experiments with wheat germ ribosomes yielded an equivalent inhibition pattern. The data suggest that the universally conserved dodecamer containing the cleavage site G2661 is located at the presumably overlapping region of the binding sites of both elongation factors.     

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.     

Further evidence for the participation of proteins S 3, S 14 and S 19 in tRNA binding to E. coli 30 S subunits

Previous studies have shown that iodination of 30 S subunits causes inactivation for both enzymatic fMet-tRNA and non-enzymatic phe-tRNA binding activities. This inactivation was shown to be due to the modification of three to five ribosomal proteins [1]. In this report the role of these proteins in tRNA binding activity has been further studied. Purified ribosomal proteins, isolated from modified subunits, are re-assembled into otherwise unmodified 30 S ribosomes and assayed for tRNA binding capacity. The presence of modified S 3, S 14 and S 19 (S 15) in the reconstituted particle results in substantial reduction of both fMet-tRNA and phe-tRNA binding activities. This reduction in tRNA binding activity does not appear to be due to an assembly defect.     

Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.     

Stoichiometric analysis of barley plastid ribosomal proteins

We analyzed the protein composition of plastid 70S ribosomes isolated from the stromal fractions of barley plastids by the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR 2D-PAGE). Intactness of the ribosomes was confirmed by the poly(U)-directed phenylalanine polymerization activity and by the reassociation capacity of the subunits into 70S ribosomes. The small and large ribosomal subunits were composed of 23 and 36 proteins, respectively. In addition, one acidic protein associated with ribosomes in low salt buffer but released in high salt buffer was found. The plastid ribosomes contained relatively larger numbers of acidic proteins than prokaryotic ribosomes. Stoichiometric analysis revealed the presence of several ribosomal proteins in low copy numbers, indicating that the ribosomes of plastids were heterogeneous. We also investigated the protein composition of plastid ribosomes from greening barley leaves and found that it did not change during greening.     

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.     

Purification and some properties of a protein binding and deacylating initiator transfer ribonucleic acid

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNA(fMet) and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNA(Met) or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8-10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNA(fMet) is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNA(fMet) and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1-2mm). The ternary complex of the factor with methionyl-tRNA(fMet) and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNA(fMet) not required for translation.     

Interaction of selectively spin-labeled 70S ribosomes with tRNAPhe. An electron paramagnetic resonance study

70S ribosomes from Escherichia coli, selectively spin labeled on the SH groups of proteins S18, S12, S21, S17, and L27, were used to study the formation of the tertiary complex ribosome-poly(U)-tRNAPhe. Most of these ribosomal proteins are located in the region of binding of tRNA. The electron paramagnetic resonance observable structural change suggests a loosening of the ribosome structure upon binding of the tRNA molecule.     

EFG-independent translocation of the mRNA:tRNA complex is promoted by modification of the ribosome with thiol-specific reagents

Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.     

High heterogeneity within the ribosomal proteins of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.Patrick Giavalisco, Daniel Wilson are contributed equally to this work.     

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA

Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA^Aspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA^Asp competed with tRNA^Aspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.     

Chemical accessibility of 18S rRNA in native ribosomal complexes: interaction sites of mRNA, tRNA and translation factors

During protein synthesis the ribosome interacts with ligands such as mRNA, tRNA and translation factors. We have studied the effect of ribosome-ligand interaction on the accessibility of 18S rRNA for single strand-specific modification in ribosomal complexes that have been assembled in vivo, i. e. native polysomes. A comparison of the modification patterns derived from programmed and non-programmed ribosomes showed that bases in the 630- and 1060-loops (530- and 790-loops in E. coli) together with two nucleotides in helices 33 and 34 were protected from chemical modification. The majority of the protected sites were homologous to sites previously suggested to be involved in mRNA and/or tRNA binding in prokaryotes and eukaryotes, implying that the interaction sites for these ligands are similar, if not identical, in naturally occurring programmed ribosomes and in in vitro assembled ribosomal complexes. Additional differences between programmed and non-programmed ribosomes were found in hairpin 8. The bases in helix 8 showed increased exposure to chemical modification in the programmed ribosomes. In addition, structural differences in helices 36 and 37 were observed between native 80S run-off ribosomes and 80S ribosomes assembled from isolated 40S and 60S subunits.     

Ribosomal proteins of mouse kidney: Normal status and during compensatory renal hypertrophy

Summary Ribosomes were isolated from normal and growing kidney and the protein complement was examined by a two-dimensional gel electrophoretic procedure. Proteins were resolved in the first dimension on the basis of charge and, in the second dimension, on the basis of molecular weight. 60S and 40S ribosomal subunits from normal kidney contained respectively 42 and 31 proteins. 80S ribosomes contained 23 proteins not found with either sub-unit. Nineteen of these proteins were removed from the ribosomes when isolated ribosomes were washed in a high salt buffer. Six proteins of the 80S ribosome corresponded to proteins associated with both sub-units. 80S ribosomal proteins were also studied during compensatory renal hypertrophy after 4-96 h of induced growth. The protein complement displayed by electrophoresis was identical to the pattern seen from normal renal cells.Abbreviations Bis-Tris [bis(2-Hydroxyethyl)imino-tris (Hydroxymethyl)methane] - MES 2(N-morpholino)ethane sulfonic acid Supported by NIH Grants AM-12769 and RR-05486 and the Damon Runyon-Walter Winchell Fund. Dr. Irwin is a fellow of the Damon Runyon-Walter Winchell Fund (DRG-51-F). Dr.Northrup is a Research Fellow in Developmental Medicine (HD00362) at Massachusetts General Hospital.     

Codon-anticodon interaction at the P site is a prerequisite for tRNA interaction with the small ribosomal subunit

The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only approximately 15% of a tRNA, namely residues 30 +/- 1 to 43 +/- 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-A crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.     

Affinity labeling of the virginiamycin S binding site on bacterial ribosome

Virginiamycin S (VS, a type B synergimycin) inhibits peptide bond synthesis in vitro and in vivo. The attachment of virginiamycin S to the large ribosomal subunit (50S) is competitively inhibited by erythromycin (Ery, a macrolide) and enhanced by virginiamycin M (VM, a type A synergimycin). We have previously shown, by fluorescence energy transfer measurements, that virginiamycin S binds at the base of the central protuberance of 50S, the putative location of peptidyltransferase domain [Di Giambattista et al. (1986) Biochemistry 25, 3540-3547]. In the present work, the ribosomal protein components at the virginiamycin S binding site were affinity labeled by the N-hydroxysuccinimide ester derivative (HSE) of this antibiotic. Evidence has been provided for (a) the association constant of HSE-ribosome complex formation being similar to that of native virginiamycin S, (b) HSE binding to ribosomes being antagonized by erythromycin and enhanced by virginiamycin M, and (c) a specific linkage of HSE with a single region of 50S, with virtually no fixation to 30S. After dissociation of covalent ribosome-HSE complexes, the resulting ribosomal proteins have been fractionated by electrophoresis and blotted to nitrocellulose, and the HSE-binding proteins have been detected by an immunoenzymometric procedure. More than 80% of label was present within a double spot corresponding to proteins L18 and L22, whose Rfs were modified by the affinity-labeling reagent. It is concluded that these proteins are components of the peptidyltransferase domain of bacterial ribosomes, for which a topographical model, including the available literature data, is proposed.     

Topography of 5.8 S rRNA in rat liver ribosomes. Identification of diethyl pyrocarbonate-reactive sites

The topography of 5.8 rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 S-28 rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gel-sequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the "AU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two -G-A-A-C- sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes.     

Binding of GDP to a ribosomal protein after elongation factor-2 dependent GTP hydrolysis.

Incubation of 80S ribosomes with a substoichiometric amount of [alpha-32P]GTP and with eEF-2 resulted in the specific labeling of one ribosomal protein which migrated very close to the position of the acidic phosphoprotein P2 from the 60S subunit in two-dimensional isofocusing-SDS gel electrophoresis. Localization of protein P2 in this electrophoretic system was ascertained by correlation with its position in the standard two-dimensional acidic-SDS gel electrophoresis after its specific phosphorylation by casein kinase II. Labeling of the ribosomal protein was dependent on the presence of eEF-2, and could be attributed to [alpha-32P]GDP binding from the results of chase experiments and HPLC identification, this binding being very likely responsible for the slight shift in the electrophoretical position of the protein. Incubation of ribosomes with tRNA(Phe) in the absence of mRNA induced the release of the bound GDP.