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Originally published In Press as doi:10.1074/jbc.M511728200 on April 18, 2006

J. Biol. Chem., Vol. 281, Issue 25, 17203-17211, June 23, 2006
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Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity*

Naomi Hirabayashi, Neuza Satomi Sato1, and Tsutomu Suzuki2

From the Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Received for publication, October 31, 2005 , and in revised form, April 11, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U({Psi})1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribosomes translate the genetic information contained in mRNAs into proteins. The large (50 S) subunit of the ribosome catalyzes the formation of a peptide bond between the aminoacyl-tRNA (aa-tRNA)3 bound to the A-site and the peptidyl-tRNA at the P-site. This peptide bond formation takes place at the peptidyltransferase center of the 50 S subunit. Codon-anticodon pairing occurs at the decoding center of the small (30 S) subunit. The aa-tRNA is delivered to the ribosome as a ternary complex of aa-tRNA, EF-Tu, and GTP. Cognate codon recognition is strictly monitored by 16 S rRNA and triggers GTP hydrolysis and dissociation of EF-Tu. This allows aa-tRNA to be accommodated by the A site of the 50 S subunit. Thus, accuracy of protein synthesis is based on the synergistic interplay of the large and small subunits of the ribosome. However, mechanistic insights into the ribosome dynamics during decoding are still rudimentary.

The intersubunit bridges of the ribosome are functional sites that are not only necessary for subunit connection but also play roles in translation. Helix 69 (H69) (position 1906-1924) is a highly conserved stem-loop in domain IV of 23 S rRNA of the bacterial 50 S subunit (Fig. 1A). In fact, each base in the loop of H69 shows more than 98% conservation in 436 bacterial rRNAs (www.rna.icmb.utexas.edu). Crystallographic studies revealed that H69 is located on the surface involved in intersubunit association with the 30 S subunit by connecting with helix 44 (h44) of 16 S rRNA forming the bridge B2a; A1912, A1913, A1914, A1918 and A1919 in H69 make contact with positions 1407-1410 and 1494-1495 in h44 and G1517 in h45 (1, 2) (see Fig. 7A). In the free 50 S subunit, H69 makes a compact structure and interacts with H71 of 23 S rRNA (3), whereas in the 70 S ribosome H69 stretches toward the small subunit and interacts with h44 of 16 S rRNA (1). The tip of H69 moves about 13.5 Å during this structural change. In addition, H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step (1). During the decoding step, aa-tRNA is brought into the A/T-site as a complex with EF-Tu/GTP (ternary complex). Cryoelectron microscopy analyses showed a kinked conformation for aa-tRNA at the A/T state (molecular spring) (4, 5). The tip of H69 makes a contact with the hinge of the kink between D- and anticodon-stems in tRNA. This interaction is supposed to facilitate the structural distortion of tRNA that enables the anticodon-stem to fit into the decoding center of 16 S rRNA. In the crystal structure of the 70 S ribosome complexed to both A- and P-site tRNAs, H69 is positioned between the two tRNAs (1). The minor groove of H69 (positions 1908-1909, 1922-1923) interacts with the minor groove of the D-stem of the P-site tRNA (positions 12-13, 25-26) (Fig. 1B), whereas the conserved loop (positions 1913-1915) of H69 makes a contact with the D-stem of A-site tRNA (positions 11-12 and 25-26) (Fig. 1B). Moreover, it has been reported that H69 interacts with various translational factors. In the post-translocational state, H69 is proximal to EF-G (6). Chemical footprinting analysis revealed that the C-terminal domain (CTD) of IF3 binds to h44 in 16 S rRNA, which is the site of bridge B2a, indicating that CTD of IF3 mimics H69 to protect subunit association during translation initiation (7). Crystallographic and cryoelectron microscopy studies showed that H69 also interacts with RF2 (8, 9), RF3 (10), RRF (11), and SmpB (12) bound to transfer-messenger RNA-EF-Tu complex. These observations indicate a pivotal role for H69 in various steps during translation.

To more precisely define the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a systematic genetic method that we named SSER (systematic selection of functional sequences by enforced replacement).4 This method allowed us to identify the residues absolutely essential for ribosomal function in Escherichia coli cells from a randomized rRNA library. The library was constructed by completely randomizing the loop sequence of H69 (Fig. 1B). The variants were then subjected to selection, and the selected variants were sequenced. The selected variants contained natural rRNA sequences from other organisms as well as unnatural but nonetheless functional sequences. The results throw new light on the nature of the bases required for H69 function. Biochemical and genetic analysis of H69 variants provide insights into the functional roles played by H69 in translation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, and Cultivation—E. coli {Delta}7rrn strain TA542 ({Delta}rrnE {Delta}rrnB {Delta}rrnA {Delta}rrnH {Delta}rrnG::cat {Delta}rrnC::cat {Delta}rrnD::cat {Delta}recA56/pTRNA66 pHKrrnC) (14) was kindly provided by Dr. Catherine L. Squires (Tufts University). The rescue plasmid pRB1014 was constructed by introducing the sacB gene and rrnB operon into pMW118 (Ampr) (Nippon gene). The plasmid pRB1024 was constructed from pMW218 (Kmr) (Nippon gene) by inserting the rrnB operon only. The plasmid pHKrrnC in strain TA542 was replaced by pRB101 to generate strain NT101, which was used as the host cell for SSER selection and B-scan analysis. Cells were grown at 37 °C in 2x Luria-Bertani medium. For solid medium, 1.5% agar was added to LB medium. Antibiotics were added at the following concentrations when required: 40 µg/ml spectinomycin (Spc), 100 µg/ml ampicillin (Amp), and 50 µg/ml kanamycin (Km). To induce plasmid replacement in NT101, 5% sucrose was added to the LB medium. NT102 is an E. coli strain series that harbors pRB102 or its derivatives instead of pRB101. The growth rates of the NT102 variants were determined by measuring the optical density at 600 nm every 15 min using a plate reader (Molecular Devices, Inc.).

Construction of the Plasmid Library of 23 S rRNA Randomized at Position H69—G1910-C1920 of H69 in 23 S rRNA (Fig. 1B) encoded in pRB102 was flipped by QuikChange site-directed mutagenesis (Stratagene) according to the manufacturer's instructions using a set of primers C1910-G1920F (5'-cggcggccctaactataagggtcctaaggtagcg-3') and C1910-G1920R (5'-ccttaggacccttatagttagggccgccgtttaccgggg-3'). This resulted in the construction of pRB102-(C1910-G1920), which was used as a template for PCR randomization to distinguish the selected variants from contamination of the template plasmid after SSER. The resulting plasmid was checked to ensure that the mutations did not confer dominant lethality by introducing it into NT101 (by the process described below). pRB102-(C1910-G1920) was hypermethylated by DNA methyltransferases, M-AluI, M-HaeIII, and M-HapII (Takara BIO, Inc.) as described4 and employed as a template for PCR randomization to reduce the background for SSER selection. In the first PCR, a set of primers complementary to sequences just outside the target sequence, gap-F (5'-ggtcctaaggtagcgaaattccttgtcggg-3') and gap-R (5'-ggccgccgtttaccggggcttcgatcaag-3'), was employed to generate gapped pRB102. This enhances the efficiency of PCR randomization and reduces the background for SSER selection. The gapped pRB102-(C1910-G1920) was then gel-purified and used as a template for PCR randomization. The N primer (5'-gctaccttaggaccgtNNNNNNNacggccgccgtttaccgggg-3') and the gap-F primer were employed to randomize seven bases in the loop of H69.4 C1910-G1920 mutations were changed back to the original sequence in this step. To obtain enough material for the randomized library, the reaction was performed in 56 tubes (total 2800 µl). After PCR, the reaction mixture was treated with DpnI (New England Biolabs) and {lambda} exonuclease (New England Biolabs) to digest the template plasmid. The products were then cleaned up with QIA-Quik (Qiagen) and checked by agarose gel electrophoresis. Unbiased distribution of four nucleotides at each randomized position was confirmed by direct sequencing of the plasmid library before selection.

SSER Selection—Detailed procedure of SSER was described in Sato et al.4 NT101 was then transformed by the randomized library and spread onto LB plates (56 plates in total) containing Spc/Km. Transformants (896 colonies) were picked to make a suspension in LB broth and then spotted onto LB plates containing Spc/Km/sucrose to force plasmid replacement. When variants showing slow growth phenotypes were picked, their resulting cell suspensions were first spotted and cultivated on LB plates containing Spc/Km (instead of being spotted directly onto LB-sucrose plates) before transfer to LB-sucrose plates. Sucrose-resistant colonies (48 colonies) (NT102 variants) were then cultured in 2x LB broth to make mini-preps of plasmids for sequencing. To check for plasmid replacement, each transformant was spotted onto two LB plates containing Spc/Amp and Spc/Km/sucrose. No growth of the spotted cells on the Amp plate demonstrated complete plasmid replacement. Functional variants were sequenced by the ABI Prism 3100 Genetic Analyzer (Applied Biosystems).

B-scan Analysis and Deletion-Insertion Mutations—To analyze the functional importance of the conserved loop of H69 (1912-1918), we replaced these positions with the other three bases by QuikChange site-directed PCR mutagenesis (Stratagene) using a set of mixed primers. Thus, if the targeted position has A, G, C, or T, it was replaced with B (G, C, and T), H (A, C, and T), D (A, G, and T) or V (A, G, and C), respectively. The primer sets employed were B1912F (5'-ccggtaaacggcggccgtBactataacggtcctaagg-3' and B1912R (5'-ccttaggaccgttatagtVacggccgccgtttaccgg-3'), B1913F (5'-cggtaaacggcggccgtaBctataacggtcctaaggtagc-3') and B1913R (5'-gctaccttaggaccgttatagVtacggccgccgtttacc-3'), D1914F (5'-ggtaaacggcggccgtaaDtataacggtcctaaggtagc-3') and D1914R (5'-gctaccttaggaccgttataHttacggccgccgtttacc-3'), V1915F (5'-gtaaacggcggccgtaacVataacggtcctaaggtagc-3') and V1915R (5'-gctaccttaggaccgttatBgttacggccgccgtttac-3'), B1916F (5'-gtaaacggcggccgtaactBtaacggtcctaaggtagc-3') and B1916R (5'-gctaccttaggaccgttaVagttacggccgccgtttac-3'), V1917F (5'-gtaaacggcggccgtaactaVaacggtcctaaggtagc-3') and V1917R (5'-gctaccttaggaccgttBtagttacggccgccgtttac-3'), B1918F (5'-gtaaacggcggccgtaactatBacggtcctaaggtagcg-3') and B1918R (5'-cgctaccttaggaccgtVatagttacggccgccgttta-3'). The primer sets for deletion and insertion mutations are {Delta}1913F (5'-cggtaaacggcggccgtactataacggtcctaaggtagc-3') and {Delta}1913R (5'-gctaccttaggaccgttatagtacggccgccgtttacc-3'), {Delta}1916F (5'-gtaaacggcggccgtaacttaacggtcctaaggtagc-3') and {Delta}1916R (5'-gctaccttaggaccgttaagttacggccgccgtttac-3'), {Delta}(1910-1920)F (5'-cggcggcctaactataaggtcctaaggtagcg-3') and {Delta}(1910-1920)R (5'-ccttaggaccttatagttaggccgccgtttaccgggg-3'), insN1913-4F (5'-ggtaaacggcggccgtaaNctataacggtcctaaggtagc-3') and insN1913-4R (5'-gctaccttaggaccgttatagNttacggccgccgtttacc-3'). As with the SSER method, the resulting pRB102-derived library bearing mutations at each position was checked by agarose gel electrophoresis and then introduced into NT101. Functional variants were selected on sucrose plates and sequenced. If the target base was essential for ribosomal function, no variants could be obtained.

Subunit Association—NT102 of each variant was cultured in 50 ml of 2x LB medium until 0.5 A600 and harvested. The cultured cells were dissolved in RBS buffer (20 mM Hepes-HCl (pH 7.6), 30 mM NH4Cl, 6 mM Mg(OAc)2, and 6 mM 2-mercaptoethanol) with 0.75 mg/ml lysozyme. After freeze-thawing by liquid N2, 200 µl of the lysate was loaded onto a 10-40% (w/v) sucrose gradient and centrifuged for 14 h at 20,000 rpm in a SW28 rotor. As for Mg2+ titration in Fig. 2B, Mg2+ concentration was adjusted with RBS buffer (6, 10, and 15 mM). Sucrose density gradient (SDG) centrifugation profiles of ribosomal subunits were monitored by using a Bio-mini UV monitor (ATTO, Japan).

Translational Fidelity Measured by beta-Galactosidase Activity—The mobile plasmid pNT3-lacZ (Ampr) (kindly provided by Dr. Akiko Nishimura) (15) was employed to construct a series of lacZ reporters for measuring the recoding activities of ribosome variants. Windows of test sequences for frameshift or read-though were introduced at the beginning of the lacZ open reading frame in pNT3-lacZ. The resultant plasmids pNT3-lacZ (+1), pNT3-lacZ (-1), pNT3-lacZ (UGA), and pNT3-lacZ (UAG) were used to measure +1 or -1 frameshift and UGA or UAG stop codon read-through, respectively. E. coli JA200 (F+ {Delta}(trpE)5 recA thr-1 leu-6 lacY thigal xyl ara mtl), which was used as a donor strain for plasmid transfer, was transformed with each reporter plasmid. Each E. coli NT102 bearing a pRB102 with a H69 variation was mated with the donor strain harboring each of the lacZ reporter plasmids transferred through the pilus during conjugation (15). For mating, precultured JA200-pNT-lacZ (10 µl) and NT102 (10 µl) were mixed and diluted in 200 µl of 2x LB without antibiotics. After incubation for 4 h at 37 °C, NT102 harboring the pNT-lacZ reporter was selected on LB plates containing Spc, Km, Amp, and 5% sucrose. Single colonies were grown to confluence, diluted (1:30) into fresh medium, and cultured at 37 °C until logarithmic phase; the cells were harvested at 0.5 A600. The beta-galactosidase assay was performed according to Miller (16). A detailed description for this method will be published elsewhere.

Preparation of Ribosomes and Elongation Factors—Ribosomes were prepared from variant strains as described (17) with slight modifications. Each E. coli strain grown up to 0.5 A600 was harvested, ground with Al2O3, then dissolved in a buffer consisting of 20 mM Hepes-KOH (pH 7.6), 30 mM NH4Cl, 10 mM Mg(Ac)2,and 6 mM 2-mercaptoethanol. The lysate was subjected to ultracentrifugation to obtain the crude ribosome. 70 S tight-coupled (TC) ribosomes were then purified from the crude ribosome preparation by 14-16 h of ultracentrifugation in a 6-38% (w/v) SDG as described (18). Recombinant elongation factors were prepared as described (19).

Poly(U)-programMed Polyphenylalanine Synthesis—In vitro translation was performed at 37 °C in 80 µl of reaction mixture consisting of 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 60 mM KCl, 6.5 mM MgCl2, 0.5 mg/ml poly(U), 0.1 mM spermine, 2.5 mM phosphoenolpyruvic acid, 2.5 unit/ml pyruvate kinase, 0.5 mM GTP, 0.6 µM E. coli EF-Tu, 0.6 µM E. coli EF-G, 0.15 µM E. coli EF-Ts, 0.075 µM 70 S ribosome, and 0.15 µM [14C]Phe-tRNAPhe. 10 µl of the reaction mixture was spotted onto filtration paper (Whatman No. 3MM) every minute. Trichloroacetic acid-insoluble product was quantified by liquid scintillation counting (ALOKA) after Phe-tRNAPhe deacylation by heating at 80 °C for 30 min. In the experiment shown in Fig. 4B, the concentration of [14C]Phe-tRNAphe ranged from 0 to 0.9 µM. Amounts of [14C]Phe incorporated in 10 µl of the reaction mixture at 6 min were plotted.

tRNA Binding to A and P Sites—For P-site tRNA binding (Fig. 5A), 12.5 pmol of 70 S ribosome, 2.5 pmol of Ac-[14C]Phe-tRNAPhe, and 2.5 µg of poly(U) in a buffer containing 50 mM Tris-HCl (pH 7.5), 6.5 mM MgCl2, 60 mM KCl, 1 mM dithiothreitol, and 0.5 mM spermine were incubated at 37 °C for 15 min. An aliquot was then spotted onto a nitrocellulose filter (Advantec) and washed with 5 ml of wash buffer (50 mM Tris-HCl (pH 7.5), 6.5 mM MgCl2, 60 mM KCl, 1 mM dithiothreitol, and 0.5 mM spermine). Ac-[14C]Phe-tRNAPhe bound on the P-site was quantified by liquid scintillation counting (ALOKA). Concerning A-site tRNA binding (Fig. 5B), the P-site of the ribosome was occupied by deacyl-tRNAPhe in a mixture (10 µl) consisting of 5 pmol of E. coli 70 S TC ribosome, 2 µg of poly(U), 10 pmol of E. coli tRNAPhe, 50 mM Tris-HCl (pH 7.5), 6.5 mM MgCl2, 60 mM KCl, 1 mM dithiothreitol, and 0.5 mM spermine, which was incubated at 37 °C for 20 min. Then 10 µl of aa-tRNA mixture consisting of 2.5 mM phosphoenolpyruvic acid, 2.5 unit/ml pyruvate kinase, 1 mM GTP, 10 pmol of [14C]Phe-tRNAPhe, and 12.5 pmol of E. coli EF-Tu was added to the ribosome mixture and incubated at 37 °C for 10 min. The following step was as described above.

Primer Extension Analysis to Detect Post-transcriptional Modifications in 23 S rRNA—Isolation of total RNA and primer extension to detect pseudouridine ({Psi}) were performed basically as described (20). Dried total RNA (4 µg) of each strain was dissolved in 30 µl of CMC solution (0.17 M N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), 7 M urea, 50 mM Bicine, and 4 mM EDTA) and incubated at 37 °C for 20 min. Stop solution (100 µl) consisting of 0.3 M NaOAc (pH 5.6) and 0.1 M EDTA was added to the reaction mixture, then 700 µl of EtOH was added to precipitate the RNA. After rinsing twice with 70% EtOH, the pellet was dissolved in 40 µl of 50 mM Na2CO3 (pH 10.4) at 37 °C for 4 h. After EtOH precipitation, it was dissolved in 10 µl of double-distilled dH2O. 5'-32P-Labeled primer, 5'-aatttcgctaccttaggaccg-3', complementary to positions 1920-1940 in 23 S rRNA was used for the primer extension. SuperScript III RNase H- reverse transcriptase (Invitrogen) was used for reverse transcription. The cDNAs resolved on 15% polyacrylamide gel containing 7 M urea was detected by fluoroimager (FLA-7000; Fujifilm).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comprehensive Genetic Selection of Functional Sequences of the H69 Loop in 23 S rRNA—To analyze the functional importance of the highly conserved loop sequence of H69 in 23 S rRNA, we employed our new genetic method (SSER).4 This system allows us to rapidly identify functional sequences in the cell among randomized sequences. Seven bases (1912-1918) in the loop of H69 in 23 S rRNA were completely randomized on pRB102 by a PCR-based method to construct a plasmid library with 16,384 sequence variations (Fig. 1B). Then we carried out large scale transformation of E. coli NT101 with each member of the library and selected for kanamycin-resistant transformants. If the incorporated plasmid had a toxic sequence that resulted in a dominant lethal pheno-type, a transformant would not be obtained. Because H69 is a critical site for ribosomal function, most of the sequences in the library must have been excluded during this step. Transformants on the kanamycin plate showed different colony sizes (data not shown), indicating that each cell contains a sequence that results in altered ribosomal activity. The kanamycin-resistant cells contain both pRB101 and pRB102 from the library. To drive plasmid replacement, each cell was picked and spotted onto selection plates containing kanamycin and sucrose. If the incorporated pRB102 plasmid had a functional sequence for ribosomal activity, it rapidly eliminated the pRB101 rescue plasmid, thus yielding sucrose-resistant cells (NT102 derivatives) because these two plasmids share the same replicon and are, thus, incompatible. Besides, if the incorporated plasmid had a non-functional or very weak functional sequence for ribosomal activity, the rescue plasmid could not be replaced by the introduced plasmid and the transformant became sensitive to sucrose due to the sacB gene.

About 900 colonies on kanamycin plates were picked and spotted onto sucrose plates. The functional sequences of the H69 loop in pRB102 from the sucrose-resistant cells (NT102) were sequenced. Despite the high conservation of H69-loop sequence, 13 functional variants were obtained (Table 1). A1912 and U1917 were completely conserved in all of the selected variants. This confirms that A1912 and U1917 are essential residues for ribosomal function. AACUAUA (variant 1), which is the original sequence of E. coli 23 S rRNA as well as the conserved sequences in bacterial, archaebacterial, and eukaryotic rRNAs, was actually selected from 16,384 sequence variations. An A1913G variation was found in variants 7 and 12. G1913 is a naturally occurring variation found in Streptomyces galbus and Streptomyces mashuensis. C1914 could be replaced with A or G, although C1914 is completely conserved in bacterial and archaebacterial rRNAs. A1915 and C1915 were selected as functional variations. In fact, A1915 and C1915 are found in rRNAs from Wolbachieae and Aerophrum (Archaea), respectively. A1916 was found to be replaceable by any of three other bases. C1916 is a naturally occurring variation found in Wolbachieae, Staphylococcus piscifermentans, and Crenarchaeota (Archaea). A1918G variation was found in variants 6, 9, and 13. G1918 is a conserved base in eukaryotic rRNAs. In addition, G1918 is a natural variation found in some bacterial and archaebacterial rRNAs. Thus, the consensus sequence of the H69 loop was ARVHNUR (Table 1).5


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  		TABLE 1
Functional sequences (1912-1918) in the H69 loop of E. coli 23 S rRNA as selected by SSER from randomized rRNA libraries Invariant or semiconserved residues selected in this study are shown in red or orange respectively. A1915 is colored blue. RNA modifications are ignored. The nomenclature used for the mixed bases obeys the rules previously established in the literature (13). Relative growth rate (RGR) was obtained by calculating the ratio of the doubling time of the mutant to that of the wild type. Consensus sequences of SSER and B-scan analysis are shown in the bottom.

 


Figure 1
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  		FIGURE 1.
Secondary structure of the domain IV in E. coli 23 S rRNA. A, analysis of the phylogenetic conservation of the E. coli domain IV of 23 S rRNA. The large subunit rRNA sequences from 436 bacteria were subjected to statistical analysis, and the individual bases in domain IV of 23 S rRNA were classified according to their degree of conservation into four categories. Positions showing more than 98% conservation are indicated by capital letters, whereas positions exhibiting 90-98% conservation are shown by lowercase letters. Positions with 80-90% or less than 80% conservation are shown by closed and open circles, respectively. Watson-Crick-type base pairs are shown by bars, whereas wobble base pairs are depicted as black dots. H69 is boxed by a dotted line. Variable regions are represented by lines with the upper and lower number of nucleotides indicated in parentheses. B, stem-loop sequence of H69 with the modified bases {Psi} and m3{Psi}. Non-canonical base pairs are shown by dotted bars. Residues (1912, 1913, 1914, 1918, and 1919) responsible for bridge B2a are circled. Residues responsible for A- and P-site tRNA binding are shown as a gray arc (1913-1915) and gray background (1908-1909 and 1922-1923), respectively. The randomized sequence for SSER is boxed. Flip mutation of G1910-C1920 to C1910-G1920 is indicated. Deletion ({Delta}) and insertion mutations tested in this study are indicated.

 
To confirm this result, we adopted another method to test the essentiality of each base of the H69 loop for ribosomal function. Each nucleotide position was replaced with any of three other bases by PCR mutagenesis using a mixed primer. In other words, if the target position was A, it was replaced with B (G, C, and T). The subsequent steps were as for the SSER method; namely, the pRB102-derived library consisting of three mutations at each position was introduced into NT101, and the functional variants were selected on sucrose plates. If a target base is essential for ribosomal function, no variants are obtained. We have named this technique B-scan analysis.4 As shown in Table 1, no variation was found at position 1912 and 1917. R1918 was also confirmed by this analysis. Although R1913 was once selected as a functional variation by SSER, A1913 was found to be replaceable by any of the three other bases. Additionally, U1914 was found to be a functional variation by this analysis. In total, 12 functional variations were obtained in this analysis, 5 of which were the same (variants 2, 3, 4, 5, and 6) as those obtained by SSER. Thus, 7 new variants were additionally obtained. The possible one-base variations in the H69-loop sequence were found to be ANNHNUR (Table 1).

The growth rate of each variant was then measured to estimate the ribosomal activity of each selected sequence (Table 1). The A1915 variant (variant 4) showed a severe growth phenotype. Variant 9 having A1915 and G1918 variations also showed a growth defect, whereas variant 6 having a single G1918 variation did not cause a growth defect. Thus, the slow growth phenotype of variant 9 is supposed to originate from A1915. However, variants 11 and 12, which contain the A1915 variation, had an improved the growth phenotype compared with that of variants 4 and 9. This result indicates that other mutations such as A1914 or C1916 might compensate for the deleterious A1915 mutation in these variants.

Because the loop size of H69 varies from 5 to 8-bases in organellar rRNAs (21), we examined the effect of deleting or inserting a base in the H69 loop on ribosomal activity (Fig. 1B). However, a one-base deletion of A1913 or A1916 or a one-base (N) insertion between A1913 and C1914 resulted in a non-functional ribosome (data not shown). In addition, deletion of the G1910-C1920 base pair in the H69-stem (Fig. 1B) also failed (data not shown). Thus, the size of the loop and the length of stem in H69 were found to be essential for ribosomal function in E. coli.

Deficiency in Ribosomal Subunit Association Among H69 Variants— The growth defect of the NT102 cell series harboring mutant rRNAs originates from the decreased functionality of each ribosome variant. Because H69 is involved in subunit association by forming the bridge B2a, we first examined subunit association in ribosome variants 9, 11, 12, and 13 using SDG centrifugation. We could not analyze variant 4 bearing a single A1915 variation due to its low viability and severe growth phenotype. At 6 mM magnesium (Mg2+) (Fig. 2, A and B), 70% of whole 50 S subunits from the wild type were incorporated into tight-coupled 70 S ribosomes (TC). In comparison, the SDG profiles of the H69 variants (9, 11, and 12) showed apparently lower TC ratios (Fig. 2A). In the case of variants 9 and 11, we observed a relative increase of the 50 S subunit. Although we cannot explain why these variants showed such phenotype, it might be a result of rescuing the weak subunit association by overproduction of the 50 S subunit. Variant 13, which has a 4-base mutation but shows normal growth rate, exhibited normal subunit association. It is known that subunit association depends on Mg2+ concentration. The SDG profile of each variant was examined at different concentrations of Mg2+ (Fig. 2B). By increasing Mg2+ concentration, the TC ratio of each variant increased to some extent (Fig. 2B). This was especially true for variant 12, which showed a significant subunit association at higher Mg2+ concentrations. However, a large fraction (60%) of the 50 S subunit from variant 9 did not associate with the 30 S subunit and was still found in the free form even at higher Mg2+ concentrations (15 mM). This result suggested that variations in the H69-loop sequence significantly influence ribosomal subunit association. Because A1915 is common to variants 9, 11, and 12, U1915 in wild type H69 appears to be important for the ribosomal subunit association that leads to normal cell growth.


Figure 2
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  		FIGURE 2.
Subunit association profiling of H69 variants. A, sucrose density gradient centrifugation profiles of H69 variants in the presence of 6 mM Mg2+. B, magnesium titration of SDG profiling. The area of each peak in the SDG profile was measured. Total 50 S is calculated by adding the area of the 50 S-peak to 63.4% of the 70 S-peak. The fraction of 50 S in the 70 S fraction can be obtained by dividing 63.4% of 70 S-peak by total 50 S. Open squares, closed squares, closed triangles, closed circles, and open circles represent wild type and variants #13, #12, #11, and #9, respectively.

 


Figure 3
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  		FIGURE 3.
Translational fidelity of H69 variants. beta-Galactosidase activities with S.D. values were measured in each H69 variant bearing four different lacZ reporter constructs; pNT3-lacZ (+1), pNT3-lacZ (-1), pNT3-lacZ (UAG), and pNT3-lacZ (UGA), which are responsible for +1 frameshift, -1 frameshift, UAG read-through, and UGA read-through, respectively. The color of each bar graph corresponds to each H69 variant indicated in the graph. The averages of three independent experiments are plotted with error bars of S.D. values. E. coli strain harboring C912G and G885U mutations in 16 S rRNA was used as a positive control for stop codon read-through (designated as error-prone, white bar).

 
Conserved H69-loop Sequences Are Important for Translational Fidelity—Because H69 plays a pivotal role in tRNA-ribosome association, we examined whether ribosomes with H69-loop mutations would perturb the accuracy of mRNA decoding by measuring the ability of ribosomes to translate engineered frameshift sites or stop codons in beta-galactosidase mRNA. For this experiment we employed the mobile plasmid system to transfer beta-galactosidase reporter constructs into the strains harboring the NT102 variants. This system uses donor strains (F+) to transfer mobile plasmids carrying lacZ reporter constructs for measuring +1 or -1 frameshift and UGA or UAG stop codon readthrough activities. The strains carrying the NT102 variants (4, 6, 9, and 11) were mated with each of the donor strains harboring each of the lacZ reporters and transformed by plasmid transfer through pilus during conjugation (15). The rapid plasmid transfer in this system enabled us to examine the translational fidelity of a vast number of strains.

An E. coli strain with error prone mutations (C912G and G885U) in 16 S rRNA, which shows significant read-through activity for UGA and UAG stop codons, was employed as a positive control (Fig. 3). The ribosome variants 4 and 9 showed strong frameshift and stop codon read-through activities (Fig. 3). Variant 6 did not show any aberrant decoding activity compared with the wild type. Variant 11 showed enhanced +1 frameshift and UGA read-through activities. This result revealed that A1915 in variants 4 and 9 is responsible for enhanced frameshift and read-through activities.

Effects of H69-loop Variations on Protein Synthesis and tRNA Binding—Next we examined the translational activity of H69 variants 9 and 11, both of which contain A1915 variation. Poly(U)-programmed polyphenylalanine synthesis by mutant and wild type ribosomes was carried out. As shown in Fig. 4A, a slow initial velocity of translation was observed for both variants 9 and 11 as compared with the wild type ribosome. Next, aa-tRNA titration experiments were employed to estimate the productivity of each variant (Fig. 4B). The productivity of each variant depended on the aa-tRNA concentration. Variant 9 required a 3-fold higher concentration of aa-tRNA to achieve the same level of productivity as the wild type. On the other hand, EF-G titration did not show any significant differences between the activities of the wild type and mutant ribosomes (data not shown). The result suggests that the slow initial velocity of H69 variants 9 and 11 is due to a requirement for high concentrations of aa-tRNA.


Figure 4
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  		FIGURE 4.
Translation activity of H69 variants. A, poly(U)-programmed polyphenylalanine synthesis of H69 variants. The concentration of [14C]Phe-tRNAPhe was 0.15 µM. Each data point represents the incorporation in the 10-µl aliquot. B,[14C]Phe-tRNAPhe titration to estimate productivity for 6 min of each variant. Each data point represents the incorporation in the 10-µl aliquot. The open square and the open and closed circles represent ribosomes from wild type and variants 9 and 11, respectively.

 
We then assessed the binding affinities of tRNAs for the mutant ribosomes using P- and A-site tRNA binding assays. Acetyl-Phe-tRNAPhe was used to bind to the poly(U)-programmed P-sites of wild type and H69 variants. As for A-site tRNA binding, a poly(U)-programmed ribosome, in which the P-site was already occupied by deacyl-tRNAPhe, was subjected to Phe-tRNAPhe binding to the A-site. No significant differences in P-site tRNA binding were observed (Fig. 5A). On the other hand, Phe-tRNAPhe bound less efficiently to the A-site of variants 9 and 11 as compared with the A-site of the wild type ribosome (Fig. 5B). This result is consistent with the decreased initial translation velocities and the high aa-tRNA concentration dependence of these variants.

Post-transcriptional Modifications in H69-loop Variants—H69 loop has two modified nucleosides, m3{Psi}1915 and {Psi}1917. It is known that the E. coli strain lacking rluD gene, which is responsible for pseudouridine formation at position 1911, 1915, and 1917, exhibited a defect in 50 S subunit assembly (22). Thus, the presence of {Psi} in the H69 loop should be important for the biogenesis of the 50 S subunit. To clarify whether these modifications occur in each variant, we analyzed m3{Psi}(m3U)1915 and {Psi}1917 using a primer extension technique (Fig. 6). Because the 3-methyl group of m3{Psi}(m3U)1915 hinders extension of the reverse transcription from the primer hybridized to 3' downstream of H69 loop, it can be detected by a normal primer extension. As shown in Fig. 6A, 3-methylpseudouridine (m3{Psi}) (or m3U) at position 1915 was clearly detected in variants 2, 5, 6, and 7, all of which have U at position 1915. In addition, after treatment with CMC (N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate), CMC-{Psi}1917 could be detected by a primer extension. The result showed that the invariant residue U1917 in all variants selected by SSER is modified to {Psi}, although its level of modification varies from variant to variant (Fig. 6A). As a control experiment (Fig. 6B), CMC-{Psi}1917 was not detected in 23 S rRNA from a strain lacking rluD gene, whereas m3U1915 was detected as described (22).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have described here a systematic genetic selection of functional sequences in the H69 loop of E. coli 23 S rRNA from 16,384 sequence variations by using the SSER method. The biggest advantage of this method is that all possible functional sequences are tested for functionality, including wild type and natural sequences found in other organisms as well as unnatural but nonetheless functional sequences that may have been excluded by the process of evolution. As such, this method permits a completely neutral genetic selection of functional sequences, as it excludes arbitrariness and experimental bias. In addition, the SSER method will even identify functional sequences that bear little or low homology to the conserved sequence, whereas other approaches using conventional site-directed mutagenesis might miss these functional sequences. Because the SSER method is based on the replacement of the rrn operon, the selection of rRNA sequences required for translating the entire proteome of E. coli is stringent. In a certain sense, the SSER mimics (albeit over a much more rapid time scale) the natural selection that occurs during evolution. This method allows us to randomize and subject any region in both the 23 S and 16 S rRNAs to selection. In addition, the selected variant is the sole ribosome species in the cell, which makes it possible to generically characterize certain phenotypes in vivo as well as permitting the homogeneous isolation of ribosome variants for in vitro biochemical characterization.

Despite the high conservation of the H69 loop, 13 functional variants, including the wild type sequence, could be obtained (Table 1). Additionally, 7 one-base variants could be obtained by B-scan analysis. Thus, we identified 20 functional variants of the H69 loop in this study. A1912 and U1917 were completely conserved in all selected variants, demonstrating that these two bases are essential for ribosomal function (Fig. 7).


Figure 5
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  		FIGURE 5.
P- and A-site tRNA binding of H69 variants. A, P-site tRNA binding of H69 variants. Bound Ac-[14C]Phe-tRNA is plotted. B, A-site tRNA binding of H69 variants. Bound [14C]Phe-tRNA is plotted. S.D. values are indicated as error bars. WT, wild type.

 


Figure 6
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  		FIGURE 6.
Primer extension analysis of 23 S rRNA from H69 variants to detect post-transcriptional modifications. A, primer extension analysis of 23 S rRNA from H69 variants (1-8) untreated (-) or treated (+) with CMC. Positions of modifications are indicated. Other variants were also analyzed (data not shown). B, primer extension analysis of 23 S rRNA from rluD deletion strain.

 
Among 20 functional variations in the H69 loop, we focused on the A1915 variation (variants 4, 9, 11, 12) for functional analysis of H69. Variant 4 has a single A1915 variation and showed a severe growth defect (relative growth rate 44%). Variant 9 bearing A1915 and G1918 variations also showed a slow growth phenotype (relative growth rate 62%) that was slightly better than that of variant 4. Because a single G1918 variation (variant 6) did not cause a growth defect, it is likely that the slow growth of variant 6 originates from the A1915 variation. Variants 11 and 12, both of which have the A1915 variation, apparently had a better growth phenotype compared with variants 4 and 9. Because variants 11 and 12 share additional A1914 and C1916 variations, these variations might have been acquired to compensate for the deleterious A1915 variation. The A1915/C1916 combination is a naturally occurring variation found in Wolbachieae rRNA, indicating that the A1915/C1916 variation may shape the functional structure of the H69 loop. To biochemically characterize ribosome variants bearing the A1915 mutation, we employed variants 9, 11, and 12. Because G1918 (the 2nd mutation of the variant 9) is an eukaryotic healthy mutation (Table 1) and variant 4 showed a similar phenotype to that of variant 9 in the beta-galactosidase reporter assay (Fig. 3), we characterized variant 9 as a representative of the A1915 variant in this study. A1915 was found to be involved in defective subunit association and weak A-site tRNA binding, which nicely explain the slow translation rate and growth defect. Because variations in the H69 loop may affect subunit association in bridge B2a, A-site tRNA binding, and translational fidelity during decoding process, it is not clear whether the growth rate of each variant is directly correlated with the lower subunit association. However, in the case of variant 13, which has a 4-base mutation in the H69 loop, it grows well and shows normal subunit association (Fig. 2, A and B). This result indicates some correlation between the growth rate and the strength of subunit association in these variants.

It is an intriguing observation that A1915 variants have low translational fidelity. During the decoding step, it is known that the tip of H69 (around position 1914) makes a direct contact with the D-stem (positions 24-25) of the A/T site tRNA (4). This interaction is considered to play an important role in the induction of the kinked conformation of aa-tRNA, which is required for correct orientation of the anticodon arm in codon-anticodon interactions in the 30 S subunit. After GTP hydrolysis and dissociation of EF-Tu from the aa-tRNA, the kinked aa-tRNA might act as a molecular spring whose relaxation would facilitate accommodation of its aminoacyl-end into the peptidyltransferase center of the 50 S subunit. It was reported that suppressor tRNATrp bearing the G24A mutation in the D-stem (Hirsh suppressor), which is the contact site of H69, enhances read-through of the UGA stop codon (23). Precise kinetic experiments revealed that the Hirsh suppressor misreads near-cognate codons by accelerating the forward rate constants of GTPase activation and accommodation into the peptidyltransferase center (24). Taken together with our observations, the specific interaction between H69 and the D-stem of tRNA might be an important factor modulating translational fidelity independently of the codon-anticodon interaction in the decoding center (24, 25).


Figure 7
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  		FIGURE 7.
Molecular interactions in bridge B2a in E. coli 70 S ribosome. A, structure of the H69 loop interacting with h44 and h45. Genetically selected essential bases (A1912 and {Psi}1917) are shown in red. R1918 is shown in orange. Other residues in H69 loop are shown in blue. Residues in h44 and h45 of 16 S rRNA involved in bridge B2a are shown in gray. H-bonds are presented by dashed lines. Coordinates were obtained from 2AWB and 2AW7. B, essential A-minor interaction in bridge B2a between H69 and h44.

 
O'Connor and Dahlberg (26) carried out a genetic selection of plasmid-encoded rRNA that promotes frameshifting of trpE91 and identified three mutations in the H69 loop, C1914U, a deletion of A1916 ({Delta}1916), and an insertion of two adenosines between A1916 and U1917 (+AA1916). These mutants showed enhanced +1 and -1 frameshift activity as well as read-through of all stop codons. More recently, Remme and co-workers (27) scanned the H69 loop by site-directed mutagenesis and analyzed the mutant ribosomes using a plasmid-encoded rRNA. They found that A1912G, A1919G, and {Psi}1917C mutations strongly inhibited translation and resulted in a severe growth phenotype and that the A1916G mutation caused defective assembly of the 50 S subunit. This approach employs plasmid-encoded mutant ribosomes in the presence of chromosomalencoded wild type rRNAs with the result that 30-40% of the ribosomes contain mutant rRNAs (27). Our SSER method is completely different from these approaches because the wild type rRNA was completely replaced in each and every case by the rRNA variant. We found that A1912G, {Psi}1917C, and {Delta}1916 were nonfunctional variations, whereas C1914U and A1916G mutations were functional variations when the wild type ribosome was replaced by each of these variants. Their results showing that {Psi}1915C and A1918G lack a clear phenotype is consistent with our data (Table 1).

Another substantial aspect of the H69 loop is the presence of the post-transcriptional modifications {Psi}1911, m3{Psi}1915, and {Psi}1917 (Fig. 1B). It is known that all of these {Psi}s are synthesized by a single pseudouridine synthase, RluD (28). The deletion strain lacking rluD gene exhibited a severe growth phenotype and a defect in 50 S subunit assembly with the appearance of immature 39 S particles (22). In addition, purified 70 S ribosomes from the deletion strain were found to be less efficient than their wild type counterparts in protein synthesis. These data indicate that the presence of {Psi} in the H69 loop is required for both the biogenesis of the 50 S subunit and for translational efficiency. From our study, m3{Psi}1915 was found to be nonessential for ribosomal function because the C1915 variant (3) selected by SSER showed a normal growth rate. In addition, it has been reported that the {Psi}1911C mutation does not affect ribosomal function (27). Thus, {Psi}sat positions 1911 and 1915 are not necessary for ribosomal function and normal cell growth. U1917 was found to be an essential residue in all variants selected by SSER method. If it is modified to {Psi} in all variants, {Psi}1917 is a candidate for the modification responsible for 50 S assembly and ribosome function. However, it was unclear how RluD might recognize sequences with changes in H69 loop. According to our primer extension analysis, {Psi} was detected at position 1917 in all variants, demonstrating that RluD can work on the H69 loop with functional variations. In the crystal structure of E. coli 70 S ribosome (2), {Psi}1917 forms a reverse Hoogsteen base pair with essential A1912 (Fig. 7B), which projects into the minor groove of base pair C1407-G1494 in 30 S subunit. {Psi}1917 might be an essential modification involved in subunit association together with A1912 as a critical function in ribosome function.

In summary, we employed a systematic genetic selection method (SSER) to identify 13 functional variants of the H69 loop out of 16,384 sequence variations. A1912 and U1917 were found to be essential residues for ribosome function. The A1915 ribosome variant showed weak subunit association, weak A site tRNA binding, and decreased translational activity, leading to a severe growth phenotype and low translational fidelity. This study revealed that H69 plays an important role in the control of translational fidelity by modulating A site tRNA binding during the decoding process.


   FOOTNOTES
 
* This work was supported by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan and by the Human Frontier Science Program (RGY23/2003) (to T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Institute Adolfo Lutz, Av. Dr. Arnaldo 355 10th floor, 01246-902 Sao Paulo, SP, Brazil. Back

2 To whom correspondence should be addressed. Tel.: 81-3-5841-8752; Fax: 81-3-3816-0106; E-mail: ts{at}chembio.t.u-tokyo.ac.jp.

3 The abbreviations used are: aa-tRNA, aminoacyl-tRNA; SSER, systematic selection of functional sequences by enforced replacement; CMC, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate); SDG, sucrose density gradient; TC, tight-coupled ribosome; {Psi}, pseudouridine; m3{Psi}, 3-methylpseudouridine; EF, elongation factor; H69, helix 69; Spc, spectinomycin; Amp, ampicillin; Km, kanamycin; Bicine, N,N-bis(2-hydroxyethyl)glycine. Back

4 N. S. Sato, N. Hirabayashi, and T. Suzuki, submitted for publication. Back

5 The nomenclature for mixed bases used in this study obey the rules previously established in the literature (13), namely, R is A or G, Y is U or C, B is all bases except A, D is all bases except C, H is all bases except G, V is all bases except U, and N is all bases (A, U, G, or C). Back


   ACKNOWLEDGMENTS
 
We are grateful to the Suzuki laboratory members, especially T. Komoda, Y. Kirino, K. Kitahara, T. Yokoyama, and M. Nagaoka, for many fruitful discussions and technical advice. We thank Dr. Catherine L. Squires (Tuft University) and Akiko Nishimura (NIG) for providing valuable strains.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity
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T. Komoda, N. S. Sato, S. S. Phelps, N. Namba, S. Joseph, and T. Suzuki
The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation
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