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Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer

2000; Springer Nature; Volume: 19; Issue: 19 Linguagem: Inglês

10.1093/emboj/19.19.5241

1460-2075

Gundo Diedrich, Christian M. T. Spahn, Ulrich Stelzl, Markus A. Schäfer, Tammy Wooten, Dmitry E. Bochkariov, Barry S. Cooperman, Robert R. Traut, Knud H. Nierhaus,

Toxin Mechanisms and Immunotoxins

Article2 October 2000free access Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer Gundo Diedrich Gundo Diedrich Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Present address: Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street FMB421, New Haven, CT, 06510 USA Search for more papers by this author Christian M. T. Spahn Christian M. T. Spahn Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Present address: Howard Hughes Medical Institute at Wadsworth Center, The Governor Nelson A. Rockefeller Empire State Plaza, PO Box 509, Albany, NY, 12201-0509 USA Search for more papers by this author Ulrich Stelzl Ulrich Stelzl Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Markus A. Schäfer Markus A. Schäfer Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Tammy Wooten Tammy Wooten Department of Chemistry, University of Pennsylvania, 231 S 34th Street, Philadelphia, PA, 19104-6323 USA Search for more papers by this author Dmitry E. Bochkariov Dmitry E. Bochkariov Department of Medical Biochemistry, University of California, Davis, CA, 95616-5224 USA Search for more papers by this author Barry S. Cooperman Barry S. Cooperman Department of Chemistry, University of Pennsylvania, 231 S 34th Street, Philadelphia, PA, 19104-6323 USA Search for more papers by this author Robert R. Traut Robert R. Traut Department of Medical Biochemistry, University of California, Davis, CA, 95616-5224 USA Search for more papers by this author Knud H. Nierhaus Corresponding Author Knud H. Nierhaus Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Gundo Diedrich Gundo Diedrich Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Present address: Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street FMB421, New Haven, CT, 06510 USA Search for more papers by this author Christian M. T. Spahn Christian M. T. Spahn Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Present address: Howard Hughes Medical Institute at Wadsworth Center, The Governor Nelson A. Rockefeller Empire State Plaza, PO Box 509, Albany, NY, 12201-0509 USA Search for more papers by this author Ulrich Stelzl Ulrich Stelzl Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Markus A. Schäfer Markus A. Schäfer Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Tammy Wooten Tammy Wooten Department of Chemistry, University of Pennsylvania, 231 S 34th Street, Philadelphia, PA, 19104-6323 USA Search for more papers by this author Dmitry E. Bochkariov Dmitry E. Bochkariov Department of Medical Biochemistry, University of California, Davis, CA, 95616-5224 USA Search for more papers by this author Barry S. Cooperman Barry S. Cooperman Department of Chemistry, University of Pennsylvania, 231 S 34th Street, Philadelphia, PA, 19104-6323 USA Search for more papers by this author Robert R. Traut Robert R. Traut Department of Medical Biochemistry, University of California, Davis, CA, 95616-5224 USA Search for more papers by this author Knud H. Nierhaus Corresponding Author Knud H. Nierhaus Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany Search for more papers by this author Author Information Gundo Diedrich1,2, Christian M. T. Spahn1,3, Ulrich Stelzl1, Markus A. Schäfer1, Tammy Wooten4, Dmitry E. Bochkariov5, Barry S. Cooperman4, Robert R. Traut5 and Knud H. Nierhaus 1 1Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany 2Present address: Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street FMB421, New Haven, CT, 06510 USA 3Present address: Howard Hughes Medical Institute at Wadsworth Center, The Governor Nelson A. Rockefeller Empire State Plaza, PO Box 509, Albany, NY, 12201-0509 USA 4Department of Chemistry, University of Pennsylvania, 231 S 34th Street, Philadelphia, PA, 19104-6323 USA 5Department of Medical Biochemistry, University of California, Davis, CA, 95616-5224 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5241-5250https://doi.org/10.1093/emboj/19.19.5241 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3′-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis. Introduction Peptide bond formation is the central enzymatic activity of the ribosome. Despite intensive experimental investigations the question of which ribosomal components form the corresponding peptidyl-transferase center (PTC) remains unanswered. In the late 1960s, Monro and co-workers showed that the 50S subunit alone is able to catalyze peptide bond formation (Monro, 1967). The 50S subunit from the Escherichia coli ribosome consists of 35 different molecules: 33 proteins and two rRNAs. Many biochemical methods have been applied to limit the peptidyl-transferase candidates to a few molecules. Components that are in close proximity to the PTC have been identified by photocrosslinking studies employing photoreactive groups attached to the CCA-end of A-, P- or E-site-bound tRNAs. Since the peptidyl-transfer reaction takes place at the ends of A- and P-site-bound tRNAs, the crosslinked molecules must be a part of or in close proximity to the catalytic center. Ribosomal components identified by this approach are the proteins L2, L15, L16, L27 and L33, and 23S rRNA (for review see Wower et al., 1993). Photoaffinity labeling with antibiotics that inhibit the peptidyl-transfer reaction provided evidence for the presence of the proteins L2, L15, L16, L18, L22, L23 and L27 as well as the central loop of domain V of 23S rRNA at or near the PTC (Cooperman et al., 1990), and photolabile oligonucleotides complementary to domain V central loop sequences place proteins L2 and L3 in the PTC vicinity (Vladimirov et al., 2000). Some of these components were excluded from catalyzing peptidyl transfer by single-omission tests: 50S subunits lacking one protein were reconstituted and tested in the puromycin reaction for peptidyl-transferase activity. It was shown that the proteins L2, L3 and L4 and 23S rRNA are essential, whereas the other proteins, as well as 5S rRNA, are dispensable (Schulze and Nierhaus, 1982; Franceschi and Nierhaus, 1990; Khaitovich and Mankin, 2000). The discovery that not only proteins but also RNA molecules can have enzymatic activity places 23S rRNA in the center of interest in the search for the peptidyl transferase. The peptidyl-transferase activity of ribosomes from Thermus aquaticus withstands treatment with proteases, SDS and phenol (Noller et al., 1992). These components destroy the native conformation of isolated proteins. However, RNA–protein interactions in ribosomes are very stable and it was not possible to remove all proteins from 23S rRNA. Eight proteins (L2, L3, L13, L15, L17, L18, L21, L22) remained stoichiometrically bound to 23S rRNA (Khaitovich et al., 1999a), and L1 and L4 were later also found (Khaitovich and Mankin, 2000). The prime candidates for the peptidyl-transferase activity are still L2, L3, L4 and 23S rRNA. Evolutionary arguments favor L2 out of the proteins, since it is one of the most conserved proteins of those universally present within the large ribosomal subunit (Müller and Wittmann-Liebold, 1997). Moreover, in studies using reconstituted subunits, mutation of the highly conserved His229 in E.coli L2 to glutamine leads to a 50S particle devoid of peptidyl-transferase activity (Cooperman et al., 1995). Related in vivo studies by Ühlein et al. (1998) also indicate the importance of this His residue in the translational activity of ribosomes. Recently, peptide bond formation was described to be catalyzed by naked mature or in vitro transcribed 23S rRNA (Nitta et al., 1998a,b), but this observation could not be reproduced by the authors and others (Khaitovich et al., 1999b; Nitta et al., 1999). Therefore, the question of whether or not 23S rRNA of the large ribosomal subunit can form peptide bonds remains unanswered. Of particular interest in this context are the results of Zhang and Cech (1998), who demonstrated that a 175 nucleotide ribozyme, selected from a random RNA library, not only can catalyze peptide bond formation but also shares secondary structure motifs with the PTC of 23S rRNA. Alternatively, the catalytic core of serine proteases has been proposed as a molecular model for the PTC, since both active centers have evolved to catalyze hydrolysis and formation of peptide bonds, respectively. The active center of serine proteases consists of the catalytic triad serine, histidine and aspartic acid, and has been developed at least twice independently by convergent evolution, as seen in the eukaryal trypsin family and the bacterial subtilisin family (Nierhaus et al., 1980; Rychlik and Cerna, 1980). There is circumstantial evidence that a histidyl residue is involved in ribosomal peptide bond formation (Nierhaus et al., 1980; Cooperman et al., 1995 and references therein). Moreover, the ribosomal protein L2 contains universally conserved seryl, histidyl and aspartyl residues. Here we report the functional effects of mutating these amino acids in E.coli L2 to test this proposal as well as the possible involvement of L2 in other ribosome functions. Results In vivo analysis In mutagenizing protein L2 we constructed two new groups of variants. Group 1, consisting of the six variants shown in Figure 1, focused on conserved histidines, serines and aspartic or glutamic acids. Group 2, consisting of the variants H14Q and H231Q, were made as controls for the previously studied H229Q. Mutations E144Q and D167N had no phenotype and are not considered further. In addition, as H229C had the same phenotype as H229A, we elected to pursue further studies only with H229A. For the Group 1 variants D83N, S177A, D228N and H229A, the gene for the ribosomal protein L2 and the mutagenized genes were cloned into the plasmid pQE-60, which plasmid adds eight additional codons for the amino acids Arg, Ser and His6 to the 3′-end of the cloned gene. Escherichia coli strain XL-1 was transformed with the plasmids. Variants H14Q, H229Q and H231Q (Group 2) were prepared from transformed E.coli cells using a conditional T7 RNA polymerase expression system as described earlier (Romero et al., 1990). DNA sequencing confirmed that the plasmid-encoded L2 genes only contain the desired mutations. Figure 1.Regions of L2 where mutations were introduced. Conserved amino acids are shaded. The mutations analyzed here are marked by an arrow. Organisms compared are E.coli (bacteria), Haloarcula marismortui (archea), Saccharomyces cerevisiae (low eukarya) and human. Download figure Download PowerPoint Growth characteristics. Growth curves were monitored for Group 1 mutants in liquid LB medium in the absence and presence of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Without IPTG all mutants tested had similar generation times, between 30 and 35 min. In the presence of 0.1 mM IPTG the doubling time of the strain expressing L2-6×H (wild-type L2 with a C-terminal His tag) was 35 min. Mutation S177A has only a minor effect on the doubling time (40 min). Mutations D83N, D228N and H229A severely reduce the growth rate, prolonging the generation time at least 8-fold (Table I). These three mutants have a dominant deleterious (lethal) phenotype. Table 1. Generation times of L2 mutants L2 mutant Generation time (min) 50S[L2] 35 50S[D83N] 280 50S[S177A] 40 50S[D228N] 320 50S[H229A] 290 Cells were grown in LB/ampicillin medium at 37°C. At 0.4 A560/ml, IPTG was added to a final concentration of 0.1 mM. At 0.8 A560/ml, cells were diluted 100-fold in fresh LB/ampicillin medium supplemented with 0.1 mM IPTG and cell growth was monitored. 50S[L2], wild type. Incorporation of Group 1 L2 mutants into 70S ribosomes and polysomes. Lysates of L2-overproducing cells were separated on a sucrose gradient (Figure 2A). The UV profiles of all mutants look similar. They show symmetrical peaks for 50S subunits, 70S ribosomes and polysomes in comparable ratios. No precursors of the 50S assembly were detected between the 30S and 50S peaks, indicating a normal assembly of the 50S subunit. 70S ribosomes and polysomes were isolated and their proteins were separated by SDS–PAGE. Overproduced, His-tagged L2 can be distinguished from wild-type L2 by its increased molecular weight. All L2 mutants are incorporated into 70S ribosomes and polysomes in similar amounts to His-tagged L2 without mutation (Figure 2B). Figure 2.Incorporation of mutagenized L2 into 70S ribosomes and polysomes. (A) Lysates from cells overproducing L2 were separated on a sucrose gradient. L2-6×H, wild-type L2 with a C-terminal His tag. (B) L2 content of 70S ribosomes and polysomes isolated from L2-overproducing cells. 70S ribosomes and polysomes shown in (A) were loaded on SDS–PAGE gels. The region of the gels between 20 and 30 kDa is shown. Download figure Download PowerPoint In vitro analysis of reconstituted 50S particles Mutagenized L2 was overproduced and purified using either the high-affinity interaction between the C-terminal stretch of six histidine residues and a Ni-NTA matrix (Group 1) or using a procedure combining streptomycin sulfate precipitation and RP-HPLC (Group 2), as described (Cooperman et al., 1995). In order to test the effect of the mutagenized L2 proteins in an in vitro translation system we constructed 50S subunits in which the wild-type L2 is replaced by the mutagenized variant. Proteins from the 50S subunit (TP50) were isolated and L2 was removed by either ion-exchange chromatography and gel filtration (Group 1) or RP-HPLC and gel filtration (Group 2) as described (Cooperman et al., 1995). The mutagenized L2, L2-depleted TP50 (TP50-L2) and rRNA were reconstituted into 50S particles that were purified from non-reconstituted material by sucrose-density centrifugation. Before testing the functional activities of the reconstituted subunits, we analyzed whether the amino acid exchange in L2 or the absence of L2 affected the 50S reconstitution or 70S formation in vitro. In vitro reconstitution of 50S subunits lacking L2. Two-dimensional gel electrophoresis revealed that the preparation TP50-L2 contained all ribosomal proteins in stoichiometric amounts, except L2, which was quantitatively absent (data not shown). TP50-L2 and total rRNAs derived from 70S ribosomes were incubated under reconstitution conditions with and without L2. At various time points of the first and the second incubation step, the formation of the three reconstitution intermediates RI50(1), RI50*(1) and RI50(2) was monitored by analyzing an aliquot of the reconstitution mixture on sucrose gradients as described previously (Dohme and Nierhaus, 1976). Figure 3 demonstrates that in the absence of L2 the formations of the intermediate particles RI50*(1) and RI50(2) were retarded, but that the final yield of 50S particles was the same as that formed in the presence of L2. The final 50S particles formed in the absence and presence of L2 had identical S values (not shown) and, except for L2, the same protein content as determined by two-dimensional (2D) gel electrophoresis. Only the amount of L16 was somewhat reduced; since the intensity of the L16 spot in the 2D gel can vary significantly even if derived from native 50S subunits, its amount cannot be estimated precisely, but is certainly >50%. Figure 3.Kinetics of the formation of the reconstitution intermediates. Particles were reconstituted from rRNA and TP50 with or without protein L2 (filled and open symbols, respectively). Aliquots were withdrawn from the reconstitution mixture during the first (left) or second (right) incubation step and subjected to a sucrose gradient centrifugation. The relative areas of the peaks of the reconstitution intermediates were determined from the A260 profile. Download figure Download PowerPoint L2 occupation in purified reconstituted 50S particles. For the Group 1 variants, purified reconstituted 50S particles were applied to SDS–PAGE in order to determine the amount of L2 incorporated (Figure 4A). Small amounts of wild-type L2 are still present in L2-depleted 50S particles (50S-L2; Figure 4B) and in all 50S containing mutagenized L2; the residual wild-type L2 stems from the 23S rRNA preparation that still contained <10% of L2. The subunits containing His-tagged L2 and the mutants D228N and H229A show L2 bands of similar intensities to those of native 50S subunits, indicating that the L2 incorporation is not hampered by the amino acid exchange or the His tag. The intensities of the mutagenized L2 bands in 50S[D83N] and 50S[S177A] are reduced to 67 and 49%, respectively. This reduced L2 occupation affects the interpretation of the in vitro assay results (see below). Figure 4.Protein content of reconstituted 50S subunits. (A) SDS–PAGE analysis of 50S subunits reconstituted with mutagenized L2 and purified by sucrose-density centrifugation. The protein equivalent of 0.3 A260 units of reconstituted particles was applied onto the gel. The intensities of the bands corresponding to mutagenized and wild-type L2 band were normalized to the intensities of the bands of L1 and the double band L3/L4 below the L2 bands, and the content of L2 is given as a percentage of the amount of L2 found in native 50S. (B) Two-dimensional gel electrophoresis of the proteins derived from native 50S subunits (left) and purified 50S particles reconstituted in the absence of L2 (right). The arrowheads indicate the spots of L2 (above) and L16 (below). In the 50S-L2 particle a residual amount of L2 of <10% is seen. Download figure Download PowerPoint For the Group 2 variants, the TP50-L2 pool used for reconstitution was virtually devoid of L2 (<2%). The amount of the L2 variant H229Q incorporated into reconstituted 50S subunits was 98% of that seen with wild-type L2, as estimated by densitometric scanning of a 2D PAGE analysis of extracted protein (Geyl et al., 1981). 70S formation of the purified 50S mutants. The association capability of the 50S Group 1 mutants was examined by incubating them with a 2-molar excess of 30S subunits under conditions that strongly favor the association of 30S and 50S subunits. 70S formation was analyzed by sucrose-density centrifugation (Figure 5). 50S subunits containing His-tagged L2 associate almost quantitatively to 70S ribosomes. In contrast, 50S subunits lacking L2 are not able to form 70S ribosomes at all. We conclude that L2 is absolutely required for the association of the 30S and 50S subunits to form the 70S ribosome. Earlier we showed that the H229Q mutation has little effect on 50S association with 30S subunits (Cooperman et al., 1995). The D228N and H229A mutations slightly impair the association (90% active), the D83N and S177A mutations are less active (70 and 50%, respectively). The reduced activities of the latter mutations can be explained by the decreased incorporation of mutagenized L2 into the 50S particles. Figure 5.Sucrose gradients of reassociated 70S ribosomes. Reconstituted 50S particle (1 A260) was incubated with a 2-molar excess of 30S subunits and loaded onto a sucrose gradient. Approximately 90% of the 50S subunits containing His-tagged L2 are associated to 70S particles. The percentages below the mutants are the area of the 70S peak compared with the 70S area of the 70S[L2-6×H] particle. Thick arrow, 70S ribosomes; intermediate arrow, 50S subunits; thin arrow, 30S subunits. Download figure Download PowerPoint Functional assays: the puromycin reaction anddipeptide formation with 50S subunits, and poly(Phe) synthesis with 70S ribosomes. Both Group 1 and Group 2 50S subunits were assayed in the puromycin reaction, whereas only Group 1 subunits were assayed for poly(Phe) synthetic activity and dipeptide formation. The His tag at the C-terminus of the recombinant L2 does not affect activities in these assays, since 50S subunits containing wild-type L2 or His-tagged L2 have the same activities (data not shown). Poly(Phe) synthesis requires 70S ribosomes and tests all reactions of the elongation cycle (A-site occupation, peptidyl transfer, translocation). The puromycin reaction and dipeptide formation are simpler systems, working with 50S subunits alone, and testing the binding of substrates to A and P sites as well as peptidyl transfer. In the puromycin reaction, fMet-tRNA (or N-AcPhe-tRNAPhe) and puromycin are substrates for P and A sites, respectively. In the case of dipeptide formation, fMet-tRNA is the P-site substrate and Phe-tRNAPhe is the A-site substrate. The results of these assays are presented in Table II. We exploited the inability of 50S-L2 to form 70S ribosomes for removal of traces of 50S particles containing L2. 50S-L2 particles were incubated under association conditions with an excess of 30S subunits, and 50S-L2 was separated from 70S and 30S by sucrose-density centrifugation. No L2 was detectable in the purified 50S-L2 by SDS–PAGE analysis and such subunits had no activity in assays of the puromycin reaction, poly(Phe) synthetic activity or dipeptide formation (Table II). Table 2. Activities (in %) of 50S mutants in functional assays relative to the activity of 50S subunits reconstituted with wild-type L2 Group 1 variants Group 2 variants D83N S177A D228N H229A 50S-L2 H229Q H14Q H231Q L2 content 74 58 98 102 0a, a, a 105b, b, b 70S association 95 86 90 88 0a, a, a 100b, b, b Poly(Phe) synthesis 53 35 64 62 0a, a, a Dipeptide formation 40 25 51 33 0a, a, a Puromycin reaction with complete tRNAs 40 33 57 8 0a, a, a 0b, b, b 105 106 AcPhe-tRNA 72 75 88 78 2 CACCA-AcPhe 65 72 Phe-tRNA 95 88 95 100 19 CACCA-Phe 84 72 CACCA-Phe 90c, c 20c, c Puromycin reaction with CACCA-AcPhe 14 4 For Group 1 variants, experimentally determined activities were corrected for the content of mutant L2 in the reconstituted 50S particles (see Figure 4), and for the presence of residual amounts of wild-type L2 (<10%) in the 23S rRNA preparation used for the reconstitution of the particles containing variant L2. For 100% values see Materials and methods. In all functional assays, native 50S subunits were included; they showed a 1.7-fold higher activity on average than the control particle reconstituted with wild-type L2. a Determined after removal of the minor fraction of 50S ribosomes still containing L2. For details see text. b Earlier results (Cooperman et al., 1995). c The stimulation of the binding of CACCA-Phe upon addition of deacylated tRNAPhe was measured. Addition of deacylated tRNAPhe leads to a 3.1-fold increase (= 100%) in CACCA-Phe binding to reconstituted 50S subunits containing L2-6×His, consistent with earlier results (Ulbrich et al., 1978). b 105 b 100 a 0 a 0 Binding to the P site: 2 72 A site: 19 72 c 20 4 Variants D83N, S177A and D228N give consistent results in all three assay systems: 40–53% activity forD83N, 25–35% for S177A and 51–64% for D228N.The inhibition seen for 50S[S177A] is likely to be due to a structural distortion of the reconstituted 50S subunit rather than a functional defect, since the corresponding mutant has a normal growth rate. Differentiated activities are obtained for the H229A mutation, with activity in the puromycin reaction (8%) being substantially lower than in dipeptide formation (33%) or poly(Phe) synthesis (62%). Furthermore, the H229Q variant is totally devoid of puromycin activity (Cooperman et al., 1995), whereas the corresponding mutation in the non-conserved His14 and less well-conserved His231 residues is without effect on the puromycin reaction. Functional assays: binding of tRNAs and tRNA fragments to various reconstituted 50S particles. To test the possibility that His229 might be involved in the binding of adenosine 76 of the A-site-bound tRNA, we investigated the relative binding affinities of tRNAs and tRNA fragments to the various reconstituted 50S particles under conditions similar to those of the puromycin reaction. The results (Table II) show that the presence of L2 is important for the binding of both P- and A-site tRNAs, with the 50S-L2 subunit having relative binding activities of 2 and 19%, respectively. In contrast, L2 is relatively unimportant for the binding of the 3′-terminal fragments CACCA-AcPhe and CACCA-Phe, which bind virtually exclusively to the P and A sites, respectively (Ulbrich et al., 1978), with the 50S-L2 subunit having relative binding activities of 72% to both sites. These contrasting effects suggest that the strong binding of tRNAs in the presence of L2 results from interactions of 50S to regions other than the 3′-ends of the tRNAs, i.e. outside the immediate PTC. However, the 3′-fragments of tRNA bind well to the 50S-L2 particles. Therefore, a puromycin reaction was performed in the presence of the fragment CACCA-AcPhe; the 50S-L2 particle has basically no and the H229A very little PTF activity (Table II). The effects of the various L2 mutations are more pronounced on P-site binding, but are in no case dramatic. Thus, binding of AcPhe-tRNA to the P site of 50S subunits containing mutated L2 is impaired by ∼25%, whereas the binding of Phe-tRNA to the A site is hardly reduced. Similarly, the H229A variant has a stronger effect (35% reduction) on CACCA-AcPhe binding than on CACCA-Phe binding (16% reduction). Cooperative effects between the binding of A- and P-site substrates of the PTC have been described. For example, addition of the P-site substrate deacylated tRNA increases the binding of the A-site substrate CACCA-Phe several-fold under fragment assay conditions (Ulbrich et al., 1978). In the experiments reported in the second last line of Table II the stimulation was 3.1-fold. Addition of deacylated tRNA increases binding of the A-site CACCA-Phe in the presence of the H229A mutation almost as strongly (2.9-fold), corresponding to a 90% stimulatory effect as compared with the wild-type control L2-6×H (Table II). This observation indicates that H229 is not involved in the fixation of the terminal A of the A-site-bound tRNA. 50S-L2 subunits show a weak stimulation of 1.4-fold in the presence of deacylated tRNA (20%; Table II). This is probably due to a poor deacylated tRNA binding in the absence of L2. Note that deacylated tRNA binds specifically to the P site under fragment assay conditions as does AcPhe-tRNA (Ulbrich et al., 1978), and, as mentioned above, P-site binding of whole tRNAs is abolished in 50S-L2 particles (see AcPhe-tRNA binding in Table II). Discussion Structural and functional analysis of 50S subunits lacking L2 Our current studies demonstrate that L2 is not essential for in vitro 50S assembly. All the reconstitution intermediates are formed, albeit more slowly than in the presence of L2 (Figure 3), and the final particle has a sedimentation constant of 50S and a full set of ribosomal L-proteins except L2, although the incorporation of L16 is somewhat reduced (Figure 4). The dependence of L16 incorporation on the presence of L2 was also observed in vivo and seems to be even stronger than during the in vitro reconstitution: 50S subunits with a deletion mutant of L2 (Thr222–Asp228) completely lack L16 and partly lack the proteins L28, L33 and L34 (Romero et al., 1990). Earlier (Cooperman et