Affinity purification of ribosomes with a lethal G2655C mutation in 23 S rRNA that affects the translocation

A method for preparation of Escherichia coli ribosomes carrying lethal mutations in 23 S rRNA was developed. The method is based on the site-directed incorporation of a streptavidin binding tag into functionally neutral sites of the 23 S rRNA and subsequent affinity chromatography. It was tested with ribosomes mutated at the 23 S rRNA position 2655 (the elongation factor (EF)-G binding site). Ribosomes carrying the lethal G2655C mutation were purified and studied in vitro. It was found in particular that this mutation confers strong inhibition of the translocation process but only moderately affects GTPase activity and binding of EF-G.     

The common and the distinctive features of the bulged-G motif based on a 1.04 A resolution RNA structure

Bulged-G motifs are ubiquitous internal RNA loops that provide specific recognition sites for proteins and RNAs. To establish the common and distinctive features of the motif we determined the structures of three variants and compared them with related structures. The variants are 27-nt mimics of the sarcin/ricin loop (SRL) from Escherichia coli 23S ribosomal RNA that is an essential part of the binding site for elongation factors (EFs). The wild-type SRL has now been determined at 1.04 Å resolution, supplementing data obtained before at 1.11 Å and allowing the first calculation of coordinate error for an RNA motif. The other two structures, having a viable (C2658U^•G2663A) or a lethal mutation (C2658G^• G2663C), were determined at 1.75 and 2.25 Å resolution, respectively. Comparisons reveal that bulged-G motifs have a common hydration and geometry, with flexible junctions at flanking structural elements. Six conserved nucleotides preserve the fold of the motif; the remaining seven to nine vary in sequence and alter contacts in both grooves. Differences between accessible functional groups of the lethal mutation and those of the viable mutation and wild-type SRL may account for the impaired elongation factor binding to ribosomes with the C2658G^•G2663C mutation and may underlie the lethal phenotype.     

Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre.

The function of the highly conserved and accessible region of domain IV of 23S rRNA (positions 1900-1981 in Escherichia coli 23S rRNA) was investigated by subjecting it to a random mutagenesis procedure that produced single-site mutations efficiently. Nine single-site mutants were selected that were recessive lethal. High levels of mutated 23S rRNA were expressed in E. coli and extracted ribosomes were investigated for their content of mutated rRNA. The peptidyl transferase activity of the ribosomes was also estimated using a newly developed method involving selective inhibition of chromosome-encoded ribosomes by clindamycin. Two of the mutants, U1940A and U1955G, yielded 50S subunits that were defective in subunit-subunit association but active in peptidyl transferase activity and five, U1926C, U1946C, U1979C, U1982A and G1984A, produced 50S subunits that were defective in both subunit-subunit interactions and peptidyl transferase activity. We infer that the large conserved rRNA region generates a complex structure that plays an essential role in maintaining and modulating subunit-subunit interactions and argue that its involvement in the peptidyl transferase centre is secondary, possibly involving the correct alignment of protein L2.     

Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes

Protein synthesis in the ribosome's large subunit occurs within an active site comprised exclusively of RNA. Mutational studies of rRNA active site residues could provide valuable insight into the mechanism of peptide bond formation, but many of these mutations cause a dominant lethal phenotype, which prevents production of the homogeneous mutant ribosomes needed for analysis. We report a general method to affinity purify in vivo assembled 50S ribosomal subunits containing lethal active site mutations via a U1A protein-binding tag inserted onto the 23S rRNA. The expected pH-dependent formation of the A2450+C2063 wobble pair has made it a potential candidate for the pH-dependent conformational change that occurs within the ribosomal active site. Using this approach, the active site A2450+C2063 pair was mutated to the isosteric, but pH-independent, G2450•U2063 wobble pair, and 50S subunits containing the mutations were affinity purified. The G•U mutation caused the adjacent A2451 to become hyper-reactive to dimethylsulfate (DMS) modification in a pH-independent manner. Furthermore, the G•U mutation decreased both the rate of peptide bond formation and the affinity of the post-translocation complex for puromycin. The reaction rate (k_pep) was reduced ~200-fold for both puromycin and the natural aminoacyl-tRNA A-site substrate. The mutations also substantially altered the pH dependence of the reaction. Mutation of this base pair has significant deleterious effects upon peptidyl transferase activity, but because G•U mutation disrupts several tertiary contacts with the wobble pair, the assignment of A2450 as the active site residue with the neutral pK_a important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data.     

Functional analysis of the invariant residue G791 of Escherichia coli 16S rRNA

The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.     

Interaction of 23S ribosomal RNA helices 89 and 91 of Escherichia coli contributes to the activity of IF2 but is insignificant for elongation factors functioning

The non-canonical base-pair C2475/G2529 joins helices 89 and 91 of the 23S rRNA in the large subunit of E. coli ribosomes. These nucleotides are located at the "crossroads" between the peptidyl transferase center, the sarcin-ricin loop and the GTPase-associated center. We probed the functional role of nucleotides C2475/G2529 by the mutations C2475G, C2475G/G2529C and deltaA2471/U2479 of 23S rRNA. All these mutations had no influence on the elongation factors activity but had different effects on the cell growth, 23S rRNA conformation and translation initiation. C2475G/G2529C and C2475G mutations led to more or less substantial decrease in IF2.GDPNP binding to the ribosomes, and IF2-assisted initiation complex formation. Ribosome-dependent GTPase activity of IF2 was enhanced by both C2475G/G2529C and C2475G mutations. Mutation deltaA2471/U2479 has no influence on IF2.GDPNP binding to the ribosome, but reduces IF2-dependent formation of initiation complex and the ribosome-dependent GTPase activity. Thus, the contact between helices 89 and 91 is important for efficient IF2 functioning in translation initiation.     

The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A.

The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escherichia coli 23S rRNAs with single mutations at positions 1067 or 1095 were expressed in vivo. Mutant ribosomes are functional in protein synthesis, although those with transversion mutations function less effectively. Antibiotics were bound under conditions where wild-type and mutant ribosomes compete in the same reaction for drug molecules; binding was analysed by allele-specific footprinting. Transversion mutations at 1067 reduce thiostrepton binding more than 1000-fold. The 1067G substitution gives a more modest decrease in thiostrepton binding. The changes at 1095 slightly, but significantly, lower the affinity of ribosomes for thiostrepton, again with the G mutation having the smallest effect. Micrococcin binding to ribosomes is reduced to a far greater extent than thiostrepton by all the 1067 and 1095 mutations. Extrapolating these results to growing cells, mutation of nucleotide 1067A confers resistance towards micrococcin and thiostrepton, while substitutions at 1095A confer micrococcin resistance, and increase tolerance towards thiostrepton. These data support an rRNA tertiary structure model in which 1067A and 1095A lie in close proximity, and are key components in the drug binding site. None of the mutations alters either the higher order rRNA structure or the binding of r-proteins. We therefore conclude that thiostrepton and micrococcin interact directly with 1067A and 1095A.     

Function of the ribosomal E-site: a mutagenesis study

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.     

Mutations in the Escherichia coli 23S rRNA increase the rate of peptidyl-tRNA dissociation from the ribosome

We have studied in vivo the phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli 50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fraction of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were under-represented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA-23S rRNA interaction has an essential role in maintaining the processivity of translation.     

Mutations in the Escherichia coli23S rRNA Increase the Rate of Peptidyl-tRNA Dissociation from the Ribosome

We have studied in vivothe phenotypes of 23S rRNA mutations G2582A, G2582U, G2583C, and U2584C, which are located at the A site of Escherichia coli50S ribosomal subunit. All mutant rRNAs incorporated into 50S ribosomal subunits. Upon sucrose gradient fractionation of cell lysates, 23S rRNAs mutated at G2582 to A and G2583 to C accumulated in the 50S and 70S fractions and were underrepresented in the polysome fraction. Induction of 23S rRNAs mutated at G2582 and G2583 lead to a drastic reduction in cell growth. In addition, mutations G2582A and G2583C reduced to one-third the total protein synthesis but not the RNA synthesis. Finally, we show that 23S rRNA mutations G2582A, G2582U, and G2583C cause a significant increase in peptidyl-tRNA drop-off from ribosomes, thereby reducing translational processivity. The results clearly show that tRNA–23S rRNA interaction has an essential role in maintaining the processivity of translation.     

Minimal functional structure of Escherichia coli 4.5 S RNA required for binding to elongation factor G

Escherichia coli cells contain abundant amounts of metabolically stable 4.5 S RNA. Consisting of 114 nucleotides, 4.5 S RNA is structurally homologous to mammalian 7 S RNA, and it plays an essential role in targeting proteins containing signal peptide to the secretory apparatus by forming an signal recognition-like particle with Ffh protein. It also binds independently to protein elongation factor G (EF-G) and functions in the translation process. This RNA contains a phylogenetically conserved RNA domain, the predicted secondary structure of which consists of a hairpin motif with two bulges. We examined the binding activity of mutants with systematic deletions to define the minimal functional interaction domain of 4.5 S RNA that interacts with EF-G. This domain consisted of 35-nucleotides extending from 36 to 70 nucleotides of mature 4.5 S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas those at A39, C40, C41, A42, G48, and G49 did not affect binding. These data suggested that the 10 nucleotides in 4.5 S RNA, which are conserved between 4.5 S RNA and 23 S rRNA, have a key role for EF-G binding. Based on the NMR-derived structure of mutant A47U, we further verified that substituting U at A47 causes striking structural changes and the loss of the symmetrical bulge. These results indicate the mechanism by which EF-G interacts with 4.5 S RNA and the importance of the bulge structure for EF-G binding.     

Gene mutations of 23S rRNA associated with clarithromycin resistance in Helicobacter pylori strains isolated from Korean patients

Although resistance of Helicobacter pylori to clarithromycin is a major cause of failure of eradication therapies, little information is available regarding gene mutations of clarithromycin-resistant primary and secondary H. pylori isolates in Korea. In the present study, we examined gene mutations of H. pylori 23S rRNA responsible for resistance to clarithromycin. DNA sequences of the 23S rRNA gene in 21 primary clarithromycin-resistant and 64 secondary clarithromycin-resistant strains were determined by PCR amplification and nucleotide sequence analyses. Two mutations of the 23S rRNA gene, A2143G and T2182C, were observed in primary clarithromycin-resistant isolates. In secondary isolates, dual mutation of A2143G+T2182C was frequently observed. In addition, A2143G+T2182C+ T2190C, A2143G+T2182C+C2195T, and A2143G+T2182C +A2223G were observed in secondary isolates. Furthermore, macrolide binding was tested on purified ribosomes isolated from T2182C or A2143C mutant strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the mutant strains. These results indicate that secondary isolates show a greater variety of 23S rRNA gene mutation types than primary isolates, and triple mutations of secondary isolates are associated with A2143G+T2182C in H. pylori isolated from Korean patients.     

Base-pairing between 23S rRNA and tRNA in the ribosomal A site

The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-p-puromycin (s4TCPm) photochemically cross-links with high efficiency and specificity to G2553 of 23S rRNA and is peptidyl transferase reactive in its cross-linked state, establishing proximity between the highly conserved 2555 loop in domain V of 23S rRNA and the universally conserved CCA end of tRNA. To test for base-pairing interactions between 23S rRNA and aminoacyl tRNA, site-directed mutations were made at the universally conserved nucleotides U2552 and G2553 of 23S rRNA in both E. coli and B. stearothermophilus ribosomal RNA and incorporated into ribosomes. Mutations at G2553 resulted in dominant growth defects in E. coli and in decreased levels of peptidyl transferase activity in vitro. Genetic analysis in vitro of U2552 and G2553 mutant ribosomes and CCA end mutant tRNA substrates identified a base-pairing interaction between C75 of aminoacyl tRNA and G2553 of 23S rRNA.     

Novel mutants of 23S RNA: characterization of functional properties.

Single point mutations corresponding to the positions G2505 and G2583 have been constructed in the gene encoding E.coli 23S rRNA. These mutations were linked to the second mutation A1067 to T, known to confer resistance to thiostrepton (1). Mutant ribosomes were analyzed in vitro for their ability to direct poly(U) dependent translation, their missence error frequency and in addition their sensitivity to peptidyltransferase inhibitors. It was evident that the mutated ribosomes had an altered dependence on [Mg2+] and an increased sensitivity to chloramphenicol during poly(U) directed poly(Phe) synthesis. In a transpeptidation assay mutated ribosomes were as sensitive to chloramphenicol as wild-type ribosomes. However, the mutant ribosomes exhibited an increased sensitivity to lincomycin. An increase in translational accuracy was attributed to the mutations at the position 2583: accuracy increased in the order G less than A less than U less than C.     

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

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

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

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

The interaction with <Emphasis Type="Italic">Escherichia coli</Emphasis> 23S rRNA helices 89 and 91 contributes to the IF2 activity but is insignificant for the functioning of the elongation factors

The noncanonical pairing of C2475 with G2529 links 23S rRNA helices 89 and 91 in the Escherichia coli ribosome. These nucleotides are at the intersection of the peptidyltransferase center, the sarcin-ricin loop, and the GTPase-associated center of the ribosome. The functional significance of C2475 and G2529 was studied using the C2475G, C2475G/G2529C, and ΔA2471/U2479 mutations of the 23S rRNA. The mutations did not change the activity of the elongation factors, but affected the cell growth rate, the 23S rRNA conformation, and the translation initiation. The C2475G and C2475G/G2529C mutations substantially affected the binding of the IF2 · GDPNP complex to the ribosome and the IF2-dependent formation of the initiation complex and increased the ribosome-stimulated GTPase activity of IF2. The Δ A2471/U2479 mutation did not affect the binding of IF2 · GDPNP to the ribosome, but influenced the IF2-dependent formation of the initiation complex and GTPase activity of IF2. The contact between helices 89 and 91 was found to be important for the efficient translation initiation catalyzed by IF2.