Polyphenylalanine Synthesis Research Articles

Mutation Analysis of the Role in Ribosomal Translocation for Loops of Domain IV of the Elongation Factor G

Seven variants of Thermus thermophilus elongation factor G (EF-G) with mutations in loops of domain IV were constructed by PCR. Point mutations Arg504 → Thr, Pro554 → Thr, or Ile534 → Asp did not affect the GTPase and translocational activities of EF-G. Similar results were obtained for mutants with tetra- or hexapeptide inserts in two loops located at the tip and two loops at the base of domain IV. Insertion of tetrapeptide Gly-Ser-Gly-Thr into loop 501–504 at the tip of domain IV dramatically reduced the activity of EF-G in poly(U)-directed polyphenylalanine synthesis on ribosomes, and halved its translocational activity. The intact conformation of loop Thr501-Gly-Gly-Arg504 was assumed to be essential for sterically perfect, efficient interaction of EF-G with the ribosome. The structural and biochemical data on the 30S subunit and EF-G were analyzed to specify the position of EF-G relative to the 30S and 50S ribosomal subunits.

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA.

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1-P2 heterodimer is required for subsequent formation of the P0(P1-P2)2 complex and its functional rRNA binding in silkworm 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.

An ATP-binding cassette transporter and two rRNA methyltransferases are involved in resistance to avilamycin in the producer organism Streptomyces viridochromogenes Tü57.

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.

Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable.

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.

Identification of a ribosomal ATPase in Escherichia coli cells

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.

TRNA aminoacylated at high pressure is a correct substrate for protein biosynthesis.

tRNA can be aminoacylated specifically with amino acids at high pressure of 6 kbar (1 bar = 1.013 atm = 0.1 MPa = 10(5) Pa) in the absence of the specific aminoacyl-tRNA synthetase and ATP. In this paper we present new evidence obtained by HPLC chromatography and TLC analysis that the esterification reaction under pressure really takes place at the 3' end of the tRNA molecule. If so, tRNA to be aminoacylated undergoes conformational changes similar to those induced with aminoacyl-tRNA synthetase. The most important finding is that aminoacyl-tRNA obtained at high pressure binds to ribosomes and participates in the synthesis of polyphenylalanine in vitro. This is the best proof of proper charging of tRNA at high pressure.

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies.

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes. These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis. Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus. Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants.

In vitro engineering using acyl-derivatized tRNAs.

AbstractIncorporation of fluorescently labeled or otherwise modified amino acids into proteins is potentially useful in a variety of ways, including monitoring the folding of the nascent protein chain, measuring posttranslational conformational changes and binding of substrates, and determining structure-function relationships. One of the most successful techniques for incorporating fluorescent amino acids into proteins has been to use aminoacyl-tRNAs in which the α-amino group has been acylated with a fluorescent moiety producing an N-acylaminoacyl-tRNA derivative (1). These derivatives have been used in then own right to study tRNA binding sites on the ribosome (2). Such derivatives, with their acylated amino groups, can only serve as donors in protein synthesis and thus can only be incorporated at the N-terminus. Nevertheless they have been very useful in studying the poly (U)1-directed synthesis of polyphenylalanine, polyserine, and polyalanine, the latter two using synthetic tRNAs whose anticodons had been changed to AAA (3). By incorporating coumarin-labeled phenylalanine or alanine at the N-terminus of such nascent chains, we were able to study by fluorescence, the interactions of the nascent chains with the ribosome as a function of chain length (4,5). More recently, using an efficient coupled transcription/translation system from Escherichia coli (6), we have incorporated coumarin-labeled methionine from tRNAf Met into several proteins. The fluorescent probe has been instrumental in studying interactions of those proteins with molecular chaperones (7).KeywordsHigh Pressure Liquid ChromatographyChloramphenicol Acetyl TransferaseSuccinimidyl EsterCircular PlasmidMercaptoacetic AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Recombinant Subunits of Mammalian Elongation Factor 1 Expressed in Escherichia coli: SUBUNIT INTERACTIONS, ELONGATION ACTIVITY, AND PHOSPHORYLATION BY PROTEIN KINASE CKII

The first step in elongation requires two different activities; elongation factor (EF)-1alpha transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1betagammadelta catalyzes exchange of GDP on EF-1alpha with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the beta, gamma, and delta subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant beta subunit migrates as a dimer and the gamma subunit as a trimer upon gel filtration, whereas the delta subunit forms a large aggregate. Complexes of betagamma, gammadelta and betagammadelta were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between beta and delta. The activity of the recombinant subunits was determined with native EF-1alpha by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant beta and delta alone stimulated the rate of elongation by 10-fold, with a ratio of 5alpha:2beta or delta. The betagammadelta complex stimulated EF-1alpha activity up to 10-fold with a ratio of 20alpha to 1betagammadelta. Phosphorylation of the beta and delta subunits alone or in betagammadelta by protein kinase CKII had no effect on the rate of elongation.

Phosphorylation of Elongation Factor 1 and Ribosomal Protein S6 by Multipotential S6 Kinase and Insulin Stimulation of Translational Elongation

Stimulation of protein synthesis in response to insulin is concomitant with increased phosphorylation of initiation factors 4B and 4G and ribosomal protein S6 (Morley, S. J., and Traugh, J. A. (1993) Biochimie 75, 985-989) and is due at least in part to multipotential S6 kinase. When elongation factor 1 (EF-1) from rabbit reticulocytes was examined as substrate for multipotential S6 kinase, up to 1 mol/mol of phosphate was incorporated into the alpha, beta, and delta subunits. Phosphorylation of EF-1 resulted in a 2-2. 6-fold stimulation of EF-1 activity, as measured by poly(U)-directed polyphenylalanine synthesis. The rate of elongation was also stimulated by approximately 2-fold with 80 S ribosomes phosphorylated on S6 by multipotential S6 kinase. When the rates of elongation in extracts from serum-fed 3T3-L1 cells and cells serum-deprived for 1.5 h were compared, a 40% decrease was observed upon serum deprivation. The addition of insulin to serum-deprived cells for 15 min stimulated elongation to a rate equivalent to that of serum-fed cells. Similar results were obtained with partially purified EF-1, with both EF-1 and ribosomes contributing to stimulation of elongation. These data are consistent with a ribosomal transit time of 3.2 min for serum-deprived cells and 1.6 min following the addition of insulin for 15 min. Taken together, the data suggest that insulin stimulation involves coordinate regulation of EF-1 and ribosomes through phosphorylation by multipotential S6 kinase.

Structure and function of nucleic acids affected by high pressure

Recently we have shown that oligodeoxynucleotides and oligoribonucleotides change their conformation at high pressure from B to Z and from A to Z respectively [Krzyzaniak et al. 1991, 1994a]. Now we are interested what is the effect of pressure on the native RNA. The structure of transfer ribonucleic acids (tRNA) and ribosomal 5S RNA undergo small conformational changes at high pressure. The changes were studied by circular dichroism (CD) but difficult to identify precisely. At high pressure conditions (6 kbar) Escherichia coli phenylalanine specific tRNA Ph~ can be aminoacylated (charged) specifically with amino acid, in absence of the specific aminoacyl tRNA synthetases and ATP [Krzyzaniak et al. 1994b]. We found that the esterification reaction at high pressure, similarly to the enzymatic one, occured at the 3'-end of the tRNA molecules. It seems plausible that a conformation oftRNA induced by the aminoacyl tRNA synthetase during enzymatic aminoacylation and that one induced at high pressure are very similar or identical. We think that the "unique" tertiary structure of tRNA creates an active centre where ester bond is formed. If so, it is obvious that a structure of the amino acid stem of tRNA determines specificity of specific tRNA with amino acid. The product of the aminoacylation reaction at high pressure, PhetRNA Phe was as good substrate as that one obtained enzymatically for in vitro polyphenylalanine synthesis in the presence of poly U. We have also shown that high pressure can catalyse elongation of short peptides. These observations have some relevance to the origin of protein synthesis. On the early stages of evolution (RNA world), RNA molecule played dual role. It could function as an enzyme and information carrier. For this reaction aminoacyl-tRNA is used as an amino acid donor. This paper and our current work strongly supports that RNA dependent amino

Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36°C in the presence of l.5 M of (NH 4) 2SO 4 100 mM of MgAc 2, 20 mM Tris-HCI (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K + and NH 4 + ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg 2+concentration present in the system.

Structure and function of 10Sa RNA: Trans-translation system

10Sa RNA is a small stable bacterial RNA in which the 5′- and 3′-end sequences are folded into a tRNA-like structure. The RNA is aminoacylatable with alanine in vitro, and it interacts with 70S ribosomes in the cell. Recently, Escherichia coli 10Sa RNA has been shown to contain the sequence-encoding tag-peptides, which are found to attach to the C-termini of truncated peptides synthesized in vivo. We have found that the E coli 10Sa RNA stimulates incorporation of the tag-specific amino acids into proteins depending on the poly(U)-directed poly-phenylalanine synthesis in the in vitro translation system. Our finding supports the ‘ trans-translation’ model proposed for the tag synthesis, in which alanyl-10Sa RNA enters the ribosome when translation stops at the 3′-end of the truncated mRNA lacking a stop codon, and translation of the tag-peptide occurs by switching the template from mRNA to 10Sa RNA. In this unique reaction, 10Sa RNA acts both as a tRNA and as an mRNA.

Dual functions of ribosome recycling factor in protein biosynthesis: Disassembling the termination complex and preventing translational errors

We summarize in this communication the data supporting the two functions of ribosome recycling factor (RRF, originally called ribosome releasing factor). The first described role involves the disassembly of the termination complex which consists of mRNA, tRNA and the ribosome bound to the mRNA at the termination codon. This process is catalyzed by two factors, elongation factor G (EF-G) and RRF. RRF stimulated protein synthesis as much as eight-fold in the in vitro lysozyme synthesis system, when ribosomes were limiting. In the absence of RRF, ribosomes remain mRNA-bound at the termination codon and translate downstream codons. In the in vitro system, the site of reinitiation is the triplet codon 3′ to the termination codon. RRF is an essential protein for bacterial life. Temperature sensitive (ts) RRF mutants were isolated and in vivo translational reinitiation due to inactivation of ts RRF was demonstrated using the β-galactosidase reporter gene placed downstream from the termination codon. A second function of RRF involves preventing errors in translation. In polyphenylalanine synthesis programmed by polyuridylic acid, misincorporation of isoleucine, leucine or a mixture of amino acids was stimulated upto 17-fold when RRF was omitted from the in vitro system. RRF did not influence the large error (10-fold increase) induced by streptomycin. This means that RRF participates not only in the disassembly of the termination complex but also in peptide elongation. Extending this concept and its conventional role for releasing ribosomes from mRNA, involvement of RRF in the reinitiation in the 3A′ system (a construct using S aureus protein A, a collaborative work with Dr Isaksson), in programmed frame shifting, in trans-translation with 10Sa RNA (collaborative work with Dr Muto), and in the reinitiation downstream from the ORF A of the IS 3 (insertion sequence of a transposon, collaborative work with Dr Sekine) are discussed on the basis of preliminary data to be published elsewhere. Finally, we review the known RRF sequences from various organisms including eukaryotes and discuss the possible mechanism for disassembly of the eukaryotic termination complex.

Effects and molecular action of ribosome-inactivating proteins on ribosomes from Streptomyces lividans

The effects of 29 type 1 and 2 type 2 ribosome-inactivating proteins (RIPS) from plants on polyuridylic acid-directed polyphenylalanine synthesis carried out by purified ribosomes from Streptomyces lividans were studied. Only dianthin 32, saporins R1 and R3, momordin I, trichokirin, Hura crepitans RIP 5 from latex, crotins 2 and 3, and PAPs C, R, and S, inhibited polyphenylalanine synthesis. Both the type 2 RIPs ricin and volkensin were ineffective on translation. The magnesium concentration affected the inhibition of translation to a considerable extent. Upon treatment with inhibitory RIPs, extraction of rRNA and further treatment with acid aniline, S. lividans ribosomes released an RNA fragment of about 130 nucleotides. The 5′ terminal sequence of this rRNA fragment was 5′-GAGGACCGGGACGGACGAACCUCUGGUGUGCCAGUUGU-3′, similar to the sequence obtained in Escherichia coh. This indicates that the most probable molecular action of these RIPs on S. lividans and E. coli ribosomes is the same: depurination of the rRNA at a site relevant to the translation mechanism and that has been highly conserved throughout evolution.

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro) 7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro) 7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro 7 sequence retains sufficient flexibility for full activity.

Comparative Analysis of Ribosome‐Associated Adenosinetriphosphatase (ATPase) from Pig Liver and the ATPase of Elongation Factor 3 from Saccharomyces cerevisiae

Elongation factor 3 (EF‐3) is a unique and essential requirement of the fungal translational machinery. Non‐fungal organisms do not have and do not require a soluble form of the third elongation factor for translation. To test whether non‐fungal organisms have a direct analog of EF‐3 incorporated in the structure of the ribosomes, a comparison of EF‐3 adenosinetriphosphatase (ATPase) with ATPases associated with pig liver ribosomes was carried out. EF‐3 function depends on ATP (GTP) hydrolysis. The hydrolytic activity of EF‐3 is enhanced by two orders of magnitude by yeast ribosomes due to an increase in the turnover rate of EF‐3. The nucleotide hydrolytic activity of EF‐3 is significantly constrained by the binding of aminoacylated tRNAphe to poly(U)‐programmed ribosomes. The translational inhibitors – neomycin and α‐sarcin suppress the ATPase activity of EF‐3. These results reflect a direct correlation between EF‐3 ATPase and the functional state of the ribosome. Four lines of evidence indicate that yeast EF‐3 ATPase is functionally distinct from pig liver ribosome associated ATPases. The kinetic parameters of ATPases from these two sources are different. Poly(U) and tRNA have no effect on the ATPase activity associated with the pig liver ribosomes. The latter activity is unaffected by the translational inhibitors neomycin and α‐sarcin. The translational activity of pig liver ribosomes is not influenced by ATP, ADP or adenosine 5′‐[β, γ‐imido]triphosphate. In an in vitro system, one can demonstrate a small but consistent stimulatory effect of yeast EF‐3 on polyphenylalanine synthesis by pig liver ribosomes only when EF‐1α is present at a limited concentration. The EF‐3 effect disappears when EF‐1α is added in a stoichiometric amount to the pig liver ribosomes. This result is in contrast to the yeast system where the ribosomes are completely dependent on EF‐3 at all concentrations of EF‐1α.