Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction*

Iwona K. WowerDagger , Jacek WowerDagger , and Robert A. Zimmermann§

From the Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003-4505

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both cold- and temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNAPhe to the A site, although the interaction of N-acetyl-Phe-tRNAPhe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Protein L27 is one of the smallest and the most basic polypeptides in the Escherichia coli ribosome (1). A combination of immune electron microscopy and protein-protein cross-linking results places L27 at the base of the central protuberance on the interface side of the 50 S subunit (2). According to in vitro studies, L27 is a late assembly protein (3) and does not have an identifiable binding site on the 23 S rRNA. Chemical and UV cross-linking studies, however, demonstrated that L27 is associated closely with domain V of the 23 S rRNA (4-6), a region that comprises part of the peptidyl transferase center (for review, see Ref. 7). The proximity of L27 to the peptidyl transferase center was also supported by affinity labeling studies with inhibitors of peptidyl transferase activity, such as chloramphenicol, carbomycin, tylosin, and spiramycin (8-11), as well as with puromycin (12), an antibiotic that mimics the aminoacyl-adenosine moiety of aminoacyl-tRNA and is a substrate for peptidyl transferase (13). Direct evidence for the presence of L27 at the peptidyl transferase center was obtained through the use of derivatives of tRNAPhe containing photoreactive azidonucleotides within the 3'-terminal ACCAOH sequence (14, 15). When bound to the ribosomal A or P site and irradiated, these probes became cross-linked predominantly to protein L27 and domain V of 23 S rRNA (15, 16).

The contribution of protein L27 to the peptidyl transferase center has been addressed by a number of studies. It has been found, for instance, that the omission of L27 during in vitro reconstitution of the 50 S subunit significantly reduces the peptidyl transferase activity of 70 S ribosomes (17, 18) and that the growth of a bacterial strain that fails to express the protein is impaired greatly (19). These findings led to the conclusion that protein L27 does not itself constitute the peptidyl transferase but that it is part of the peptidyl transferase center and is necessary for efficient peptide bond formation. To assess further the role of L27 in the bacterial ribosome, we have substituted rpmA gene, which encodes this protein, with a kanamycin resistance (Kanr) marker. The resulting strain exhibited a severe growth defect and accumulated a precursor to the 50 S particle which was deficient in several ribosomal proteins, demonstrating that L27 is important for subunit assembly in vivo. Ribosomes lacking protein L27 were also impaired in peptidyl transferase activity. Our analysis suggests that this defect is caused by the reduced affinity of aminoacyl-tRNA for the ribosomal A site.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- All restriction enzymes were purchased from New England Biolabs. Plasmid pEE, plasmid pMT101, and E. coli strain MS367 were gifts of Drs. K. Isono, P. Schimmel, and S. Brown, respectively. [14C]tRNAPhe, [14C]Phe-tRNAPhe, Ac[14C]Phe-tRNAPhe,1 and fractionated poly(U) were kindly provided by Dr. S. V. Kirillov. The sources of other enzymes, radioactive compounds, and biological materials were as described (14, 20).

Construction of the Delta rpmA::kan Strain-- Bacterial strains and plasmids used in this study are listed in Table I. E. coli strain XL1-B was the host for the purification and maintenance of newly constructed plasmids. Liquid cultures and plates consisted of 2 × YT medium containing antibiotics as needed: 50 µg/ml kanamycin, 200 µg/ml ampicillin, and 30 µg/ml chloramphenicol. All recombinant DNA and gene transfer procedures were performed by standard techniques (25, 26). Replacement of the rpmA gene with the kanamycin resistance marker was carried out by the method of Hamilton et al. (27). E. coli strain LG90 was first transformed with pTSkan and pUCrpmA plasmids. Logarithmically growing recipients were spread on prewarmed plates containing kanamycin, ampicillin, and 1 mM isopropyl-1-thio-beta -D-galactopyranoside (IPTG) and incubated at 43 °C for 2 days. Colonies resistant to both antibiotics were then subjected to three cycles of growth at 30 °C in liquid medium with ampicillin and IPTG, but without kanamycin, to promote the resolution of cointegrates. Log phase cells from the final 30 °C culture were diluted 1,000-fold and incubated for 8 h at 43 °C in the absence of kanamycin to destroy the resolved pTS plasmid. The cells were then spread on plates containing kanamycin, ampicillin, and IPTG and incubated for 2-3 days at 43 °C. Colonies resistant to kanamycin but susceptible to chloramphenicol at both 30 and 43 °C were tested by Southern blot analysis for the replacement of the chromosomal rpmA gene by kan (see Fig. 1). Transduction with bacteriophage P1 was used to transfer the kan construct into LG90 cells lacking plasmid pUCrpmA. The resulting strain, IW312 (rec+), was mated with MS367 to create strain IW326 (rec-).

                              
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Table I
Bacterial strains and plasmids used in the study

Analysis of Ribosomes and Ribosomal Proteins-- The distribution of polysomes and ribosomal particles from logarithmically growing cells was analyzed by sucrose gradient centrifugation (14). Proteins were isolated from ribosomes or ribosomal particles by acetic acid extraction and fractionated by two-dimensional PAGE (28).

Enzymatic Binding of Phe-tRNAPhe to the Ribosomal A Site-- To block the ribosomal P site, 10 pmol of 70 S ribosomes was incubated with 13 µg of poly(U) and 13 pmol of tRNAPhe for 10 min at 37 °C in 10 µl of TMA buffer (50 mM Tris-Cl, pH 7.6, 50 mM NH4Cl, 10 mM MgCl2) containing 1 mM dithiothreitol (TMA/SH). Ternary complexes were then prepared by adding 10 pmol of EF-Tu and 10 pmol of [14C]Phe-tRNAPhe (1,400 dpm/pmol) to 40 µl of a solution containing 0.58 mM GMP-PNP, 0.5 mM phosphoenol pyruvate, and 20 µg/ml pyruvate kinase in TMA/SH buffer. After 10-fold dilution, the ternary complexes were added to the tRNA-poly(U)-ribosome complexes, and the mixtures were incubated on ice. Aliquots were withdrawn at different time intervals, and the amount of [14C]Phe-tRNAPhe bound was measured by filter retention (29).

Puromycin Reaction-- Ac[14C]Phe-tRNAPhe was bound to the P site of poly(U)-programmed ribosomes (30). Reaction mixtures contained 1 µM 70 S ribosomes, 0.5 µM Ac[14C]Phe-tRNAPhe (1,400 dpm/pmol), and 1 mg/ml poly(U) in dithiothreitol (TMA/SH) buffer and were incubated for 15 min at 37 °C. After measuring the amount of tRNA bound to the ribosomes, aliquots of the complex were incubated in the presence of 0.1-1 mM puromycin for various times at 0 or 37 °C, and the initial rates of Ac[14C]Phe-puromycin formation were determined (31).

Cross-linking of Photoreactive tRNAPhe Derivatives to Ribosomes-- 32P-Labeled [2N3A76]tRNAPhe was prepared, aminoacylated, and acetylated (13, 32). Non-aminoacylated [32P][2N3A76]tRNAPhe and Ac[14C]Phe-[32P][2N3A76]tRNAPhe were purified by high performance liquid chromatography on a C4 reverse phase column (33). Photoreactive tRNAPhe dervatives were bound to ribosomes by incubating 0.5 µM tRNAPhe derivative with 1 µM 70 S ribosomes and 1.3 mg/ml poly(U) in TMA buffer for 15 min at 37 °C. Complexes were then irradiated for 2 min at 4 °C with 300-nm UV light (14). Labeled ribosomal components were analyzed as described (14, 15).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Deletion of the rpmA Gene from the E. coli Chromosome-- The rpmA gene, which encodes ribosomal protein L27, was deleted from the chromosome of E. coli strain LG90 and replaced by a kanamycin resistance marker as described under "Experimental Procedures." Southern blots demonstrated that the 6.1-kilobase BglII fragment encompassing the rplU-rpmA operon was substituted by a 7.5-kilobase fragment that lacked the KpnI restriction site present in the rpmA gene (Fig. 1). In addition, the latter fragment hybridized with a probe for kan but not rpmA. The kan construct was used to generate mutant strains IW312 and IW326, which carry the L27 deletion in rec+ and rec- backgrounds, respectively (Table I). Two-dimensional PAGE of ribosomal proteins from LG90 and IW326 showed that the mutant ribosomes lack only protein L27 (Fig. 2), and an immunoprecipitation test with anti-L27 antibodies confirmed the absence of L27 from the mutant cell extract (not shown).


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Fig. 1.   Replacement of the rpmA gene by kan. Panel A, strategy of gene replacement. The Bsu36I-AgeI fragment of the E. coli chromosome encoding rpmA was replaced with the BamHI-NheI fragment of pACYC177 carrying kan (see "Experimental Procedures"). The E. coli map is from Ref. 23. Restriction sites are: A, AgeI; Ba, BamHI; Bg, BglII; Bs, Bsu36I; H, HindIII; K, KpnI; Nh, NheI; P, PstI. Panel B, Southern blot analysis of chromosomal DNA from wild-type (LG90) and mutant (IW312) strains. Hybridization probes, shown as black bars in panel A, correspond to the PstI-Bsu36I fragment upstream from the rpmA gene (probe A), the HindII-XhoI fragment of pACYC177 including most of the kan gene (probe B), and the Bsu36I-AgeI fragment spanning the rpmA gene (probe C). The following restriction digests were used: lane 1, BglII; lane 2, BglII and PstI; lane 3, BglII and KpnI.


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Fig. 2.   Two-dimensional polyacrylamide gel analysis of ribosomal proteins from wild-type and L27-lacking E. coli. Proteins were extracted from 10 A260 units of purified 70 S ribosomes from strains LG90 (left) and IW326 (right) and separated by acidic two-dimensional PAGE (28).

Phenotype of the L27-deletion Strain-- The growth of strains LG90, IW312 and IW326 was compared at different temperatures (Table II). The wild-type strain, LG90, grew rapidly under all conditions tested. In contrast, strains IW312 and IW326 were impaired severely; at 37 °C, small colonies appeared on solid medium only after 2-3 days, and their doubling times in liquid culture were five to six times greater than that of LG90. When strains IW312 and IW326 were streaked on agar plates and incubated at 25 °C or 43 °C for up to 2 weeks, only a trace of growth was visible at the beginning of each streak. Similarly, there was no measurable growth in liquid culture at these temperatures. Thus, deletion of the rpmA gene renders the cells both cold- and heat-sensitive. However, because cells that failed to grow at 25 and 43 °C reinitiated growth upon transfer to 37 °C, the absence of L27 appears to be bacteriostatic rather than bacteriocidal at the two temperature extremes.

                              
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Table II
Growth characteristics of wild-type and mutant E. coli strains

The impaired growth of strains IW312 and IW326 was largely alleviated when plasmids carrying a functional rpmA gene were introduced into the mutant strain. The presence of plasmids pEE and pUCrpmA both led to a significant increase in cell growth rate at 37 °C and abolished cold and temperature sensitivity, whereas the corresponding parental plasmids lacking rpmA, pBR322, and pUC119, as well as a plasmid lacking the 5'-terminal half of the rpmA gene, pEEDelta BK, did not (Table II). The faster growth of cells harboring pEE as opposed to pUCrpmA is attributable to the more efficient expression of L27 in the former strain as demonstrated by specific immunoprecipitation of the protein from cell extracts (not shown).

Assembly of Ribosomal Particles Lacking L27 in Vivo-- The distribution of ribosomes in lysates of log phase LG90, IW326, and IW326/pEE cells was examined by sucrose gradient centrifugation. As shown in Fig. 3A, panel a, there is little variation in the polysome profiles of the three strains, although there is a significant difference in the ratio of 70 S monosomes to 50 S and 30 S subunits. Lysates of the wild-type strain contained a large 70 S peak with very few 50 S and 30 S particles, whereas lysates of the L27-deletion strain contained more ribosomal subunits than 70 S monosomes. In the latter case, longer centrifugation times revealed the presence of particles sedimenting at approximately 40 S in addition to the 30 S and 50 S subunits (not shown). In contrast, lysates obtained from strain IW326/pEE, in which the chromosomal deletion was complemented by a plasmid-borne copy of the rpmA gene, exhibited a ratio of 70 S ribosomes to 50 S and 30 S subunits resembling that of the wild-type strain. This observation demonstrates that protein L27 is required for efficient assembly of the 50 S subunit.


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Fig. 3.   Distribution of ribosomes, ribosomal subunits, and ribosomal RNA in wild-type and mutant bacteria. A, sucrose gradient analysis of ribosomes from strains LG90 (wild type), IW326 (Delta L27), and IW326/pEE. Panel a, crude cell lysates were fractionated on 14-40% sucrose gradients containing 10 mM Mg2+. Panel b, ribosomal subunits were resuspended from 100,000 × g ribosome pellets and centrifuged on 10-30% sucrose gradients containing 0.3 mM Mg2+. B, electrophoretic analysis of rRNA from ribosomal subunits fractionated on sucrose gradients as in panel b above. RNA was extracted with phenol/chloroform from 0.5 to 1.0 A260 units of ribosomal particles from LG90 (left) and IW326 (right) and separated by electrophoresis on a 0.7% agarose gel in TBE buffer. Lanes 1, 2, and 3 correspond to 50, 40, and 30 S fractions, respectively.

To characterize the 40 S particles present in lysates of strain IW326, crude ribosomes prepared in 10 mM Mg2+ were analyzed on sucrose gradients containing 0.3 mM Mg2+ (Fig. 3A, panel b). Whereas the sample isolated from the wild-type strain contained 50 S and 30 S subunits in the expected 1:1 ratio, the L27-deletion strain yielded three components sedimenting at approximately 50, 40, and 30 S. As evident from the figure, the 30 S peak was considerably more prominent than the 50 S peak. In the sample obtained from IW326/pEE, the presence of extrachromosomal copies of the rpmA gene led to a restoration of the typical 50 S to 30 S ratio of 2:1.

Peak fractions from the IW326 lysate were analyzed for RNA and protein content (Figs. 3B and 4). The 50 S fraction contained 23 S rRNA and all of the 50 S subunit proteins except L27. The 40 S fraction also contained 23 S rRNA, suggesting that it is a precursor to the large subunit. Two-dimensional PAGE analysis showed that large subunit proteins L16, L20, and L21 were missing from the 40 S particle in addition to protein L27. A pulse-chase experiment established that the 40 S particles mature to 50 S subunits and are therefore true intermediates in 50 S subunit assembly (not shown). Although the 30 S subunit fraction from the wild-type lysate contained mostly 16 S rRNA and was contaminated by only a small amount of 23 S rRNA, the corresponding fraction from the mutant lysate contained 23 S and 16 S rRNA in a 1:1 ratio (Fig. 3B). Therefore, the 30 S fraction appeared to contain another, smaller precursor to the 50 S subunit in addition to the 40 S particle. We conclude that protein L27 strongly affects the kinetics of 50 S subunit assembly in vivo as the absence of L27 leads to the accumulation of partially assembled precursor particles in addition to active 50 S subunits. This bottleneck in 50 S subunit maturation is very likely to contribute to the slow growth phenotype of the L27-deletion strains.


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Fig. 4.   Two-dimensional polyacrylamide gel analysis of ribosomal proteins extracted from 50 S subunits and 40 S precursor particles isolated from strain IW326. Ribosomal particles were purified on sucrose gradients in 0.3 mM Mg2+ as in Fig. 3A, panel b. Protein samples were separated by two-dimensional PAGE as in Fig. 2.

Activity of Ribosomes Lacking L27 in Vitro-- To determine the effects of L27 deletion on ribosome function, we compared wild-type and mutant ribosomes with respect to their ability to interact with tRNA and to carry out the puromycin reaction in vitro. Both types of ribosomes were first assayed for their capacity to bind AcPhe-tRNAPhe and Phe-tRNAPhe to the ribosomal P and A sites, respectively. Approximately 60% of the ribosomes from the two strains were active in binding AcPhe-tRNAPhe to the P site, and no differences in the kinetics of binding were noted (not shown). On the other hand, the rate of enzymatic binding of Phe-tRNAPhe to the A site of mutant ribosomes was slower than that to wild-type ribosomes at both 0 and 37 °C (Fig. 5). In addition, when the P site was filled with AcPhe-tRNAPhe, the L27-lacking ribosomes bound only 0.6 pmol of Phe-tRNAPhe/pmol of active ribosomes, whereas wild-type particles could bind more than 0.8 pmol of Phe-tRNAPhe/pmol of active ribosomes.


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Fig. 5.   Kinetics of EF-Tu-directed binding of Phe-tRNAPhe to wild-type and L27-lacking ribosomes. Enzymatic binding of [14C]Phe-tRNAPhe to poly(U)-programmed ribosomes whose P site had been filled previously with tRNAPhe is shown (see "Experimental Procedures"). Results are expressed as pmol of [14C]Phe-tRNAPhe bound per pmol of 70 S ribosomes from strains LG90 (open circle ) and IW326 (bullet ).

The peptidyl transferase activity of wild-type and L27-lacking ribosomes was assessed by monitoring the puromycin reaction at 0 and 37 °C as shown in Fig. 6. The maximum reaction velocities for wild-type ribosomes determined from the plot are 0.625 min-1 at 37 °C and 0.033 min-1 at 0 °C. The corresponding values for the mutant ribosomes are 0.192 min-1 at 37 °C and 0.008 min-1 at 0 °C, or three to four times less than for the wild-type ribosomes. Because the puromycin reaction is a direct measure of peptidyl transferase activity, the difference in reaction kinetics between the two kinds of ribosomes indicates that the L27-lacking ribosomes are defective in peptidyl transferase activity as well as in 50 S subunit assembly.


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Fig. 6.   Peptidyl transferase activity of wild-type and L27-lacking ribosomes. Double-reciprocal plots of the velocity of the puromycin reaction were constructed from data obtained as described under "Experimental Procedures." Ribosomes were from strains LG90 (open circle ) and IW326 (bullet ). Although the Km values for the puromycin reaction are the same for LG 90 and IW326 ribosomes at 37 °C, they differ markedly at 0 °C, suggesting a weaker interaction between the antibiotic and the A site of the mutant ribosomes at the lower temperature.

Cross-linking of Photoreactive tRNA Derivatives to Ribosomes Lacking L27-- Our previous studies have shown that a photoreactive derivative of tRNAPhe containing 2-azidoadenosine at the 3' or acceptor end labels both protein L27 and 23 S rRNA when bound and cross-linked from the ribosomal A and P sites (15, 16). In the present work, we have used the same tRNA derivative to determine how the absence of L27 affects the way in which the 3' end of tRNA interacts with the ribosome. [2N3A76]tRNAPhe and its peptidyl analog AcPhe-[2N3A76]tRNAPhe were bound to the P site of poly(U)-programmed wild-type and mutant ribosomes. P site binding was confirmed in each case through the use of edeine, an antibiotic that specifically interferes with tRNA-ribosome interaction at the P site (34, 35), and by the reaction of AcPhe-[2N3A76]tRNAPhe with puromycin (29, 36). Upon irradiation of the tRNA-ribosome complexes, 15-30% of the noncovalently bound tRNA became covalently linked to both types of ribosomes, although the pattern of labeling differed markedly (Table III). In wild-type ribosomes, AcPhe-[2N3A76]tRNAPhe was cross-linked to protein L27 and two specific sequences within domain V of the 23 S rRNA as reported earlier (14, 16). In ribosomes lacking L27, however, all of the label was associated with the 23 S rRNA, although the cross-linked sequences were identical to those in wild-type ribosomes. These results indicate that the 3' ends of the peptidyl-tRNA analog adopt a similar orientation relative to the 23 S rRNA regardless of the presence or absence of L27. A somewhat different distribution of cross-links was observed in experiments with non-aminoacylated [2N3A76]tRNAPhe; in wild-type ribosomes, the label was again associated with L27 and 23 S rRNA, whereas in mutant ribosomes, L33 was labeled in addition to 23 S rRNA. Because the labeling of L33 is primarily attributable to E site-bound [2N3A76]tRNAPhe in wild-type ribosomes (37), the present findings suggest that the 3' end of deacylated tRNA is not fixed to a unique site in the mutant ribosomes.

                              
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Table III
Cross-linking of AcPhe-[2N3A76]tRNAPhe and [2N3A76]tRNAPhe to the P site of wild-type and mutant ribosomes
Irradiated [32P]tRNA-ribosome complexes were dissociated into subunits by centrifugation through 10-30% sucrose gradients, and covalent tRNA-50 S subunit cross-linking was estimated from radioactivity cosedimenting with the 50 S peak. The percentage of tRNA cross-linked to 50 S ribosomal proteins and rRNA was measured from the distribution of 32P-labeled material on SDS-urea polyacrylamide gels. Covalent [32P]tRNA-50 S subunit complexes were digested with RNase T1 and analyzed by SDS-PAGE. The percentage of tRNA cross-linking to ribosomal proteins L27 and L33 was estimated from the distribution of 32P label between these two proteins.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Protein L27 has been identified as a component of the peptidyl transferase center of the E. coli ribosome by numerous biochemical and genetic studies (for reviews, see Refs. 38 and 39). We have shown, for instance, that L27 is the main protein cross-linked from 2- or 8-azidoadenosine at positions 73 and 76 of tRNAPhe at the P and A sites (14-16). Because the azidoadenosines give rise to very short cross-links, on the order of 3-4 Å, specific labeling of L27 by both P and A site-bound tRNAs is in accord with the expected juxtaposition of their 3'-terminal adenosines during peptidyl transfer. To characterize further the functional role of L27 in translation, we constructed a strain of E. coli from which the L27 gene was deleted. We show here that the mutant bacteria grow five times more slowly than wild-type E. coli and are both cold- and temperature-sensitive. The mutant phenotype is reversed upon expression of protein L27 from a plasmid, however, demonstrating that the impaired growth of the deletion strain results from the lack of protein L27. We have traced the molecular basis of growth impairment to interrelated defects in the assembly and function of the 50 S ribosomal subunit.

Logarithmically growing cells of the rpmA deletion strain accumulate 40 S precursors to the 50 S subunit which lack proteins L16, L20, and L21 in addition to L27. Pulse-chase experiments showed that the 40 S particles eventually mature to 50 S subunits even in the absence of L27 and subsequently are incorporated into 70 S monosomes and polysomes. This observation shows for the first time that L27 plays a role in the assembly of E. coli ribosomes within the cell and indicates that there are significant differences between 50 S subunit assembly in vivo and in vitro. The absence of L16, L20, and L21 from the 40 S particles implies that the assembly of these proteins is dependent on L27 in vivo. By contrast, in vitro reconstitution experiments indicate that L27 is a late assembly protein and therefore not expected to influence assembly of the 50 S particles. In particular, the in vitro assembly map does not predict any assembly linkage between protein L27 and proteins L16, L20, and L21, although L27 has been cross-linked to L16 in the mature 50 S subunit (40, 41) and is a neighbor of L16 according to immunoelectron microscopy studies (42). Furthermore, although L20 promotes the incorporation of L21, neither one depends upon nor stimulates L27 binding in vitro (3). Moreover, protein L20 is considered as a primary assembly protein with a binding site in the 5' third of the 23 S RNA (43), whereas protein L27, which appears to lack an independent RNA binding site, has been cross-linked to three sites in domain V of the 23 S rRNA (4-6). The presence of protein L30 in 40 S precursor particles is also unexpected because this protein does not assemble into the 50 S subunit in vitro without proteins L20 and L21. However, the early assembly of L27 in vivo is supported by the observation that a 32 S precursor found in wild-type cells contains protein L27 along with 16 other ribosomal proteins, including L20 and L21 (44). It is of interest in this connection that protein L21 is encoded by the same operon as L27 (45). Although the mechanism by which this operon is regulated has not been elucidated, deletion of the L27 gene does not appear to reduce the synthesis of L21.

It has been found recently that a 371-amino acid protein from the large ribosomal subunit of yeast mitochondria, Rml27p, contains an amino-terminal domain of 84 residues which is homologous to E. coli L27 as well as a carboxyl-terminal domain unrelated to any other known ribosomal protein (46). Deletion analysis revealed that this protein is essential for the structural and functional integrity of the yeast mitochondrial ribosome. Several amino acid substitutions in conserved positions of the L27-like domain led to cold- and temperature-sensitive defects in the assembly of the mitochondrial large ribosomal subunit and the accumulation of more slowly sedimenting particles containing large subunit rRNA. These striking parallels to the properties of L27-lacking E. coli provide further evidence that this protein contributes to the assembly of the large ribosomal subunit, a role that has apparently been conserved in the evolution of diverse genetic systems.

In addition to defects in 50 S subunit assembly, we have found that the absence of protein L27 leads to the impairment of peptidyl transferase activity. This defect appears to result from a decrease in the ability of the mutant ribosomes to facilitate EF-Tu-dependent binding of aminoacyl-tRNA to the A site. Earlier photochemical cross-linking studies have shown that the 3'-terminal adenosine of A site-bound Phe-[2N3A76]tRNAPhe is in close proximity to protein L27 (15). Our present findings suggest that L27 is not only in proximity to the 3' end of the A site tRNA but that there is a functional interaction between them which contributes to tRNA binding. This proposal is supported by our observation that removal of L27 weakens the interaction of puromycin, an analog of the 3' end of aminoacyl-tRNA (13), with the ribosomal A site. In contrast, the rate and extent of binding of a peptidyl-tRNA analog, AcPhe-tRNAPhe, to the P site were not perturbed.

We conclude from the results reported here that protein L27 plays a role in mediating the proper placement of the 3' end of A site tRNA at the peptidyl transferase center and that it may also influence the interaction of the 3' end of deacylated tRNA with the ribosome after peptidyl transfer. These inferences are consistent with several properties of the mutant ribosomes, including the lower rate of peptidyl transfer, the reduced enzymatic binding of aminoacyl-tRNA to the A site, and the observation that the 3' end of deacylated tRNA can be cross-linked to L33, a protein component of the ribosomal E site. Because L27 is an unusually basic protein (1), its function may be to screen the negative charge of the tRNA molecules from that of the 23 S rRNA during the peptidyl transferase reaction.

    ACKNOWLEDGEMENTS

We are indebted to Drs. K. Isono, P. Schimmel, S. Brown, and S. V. Kirillov for providing bacterial strains, plasmids, and materials used in this work, and to Dr. S. V. Kirillov for much useful advice and discussion.

    FOOTNOTES

* This research was supported by National Institutes of Health Grant GM22807.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Animal and Dairy Sciences, Auburn University, Auburn, AL 36849.

§ To whom correspondence should be addressed. Tel.: 413-545-0936; Fax: 413-545-3291; E-mail: zimmermann{at}biochem.umass.edu.

1 The abbreviations used are: AcPhe-tRNAPhe, N-acetylphenylalanyl-tRNAPhe; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; [2N3A76]tRNAPhe, tRNAPhe derivative containing 2-azidoadenosine at position 76; GMP-PNP, guanosine 5'-(beta ,gamma -imino)triphosphate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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