Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non–E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Δ7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9–99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself.     

Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation

To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 × 10⁻⁹). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 μg of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 ± 1.1 × 10⁻⁶, n = 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia.     

Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates

Background Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT‐PCR) was evaluated in an inter‐laboratory comparison in three different German blood services. Methods Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10³–4·5 × 10⁸ CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. Results The inter‐laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT‐PCR‐screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non‐detected positive samples were below the assay’s detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10⁵ and 2·10 × 10⁷ CFU/ml, respectively, K. pneumoniae: 4·79 × 10⁶ CFU/ml, S. aureus: 6·03 × 10⁵ CFU/ml). All rapid screening methods revealed no false‐positive results. Conclusions Both BactiFlow and 23S rRNA RT‐PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram‐negative strains.     

Isolation and identification of multidrug-resistant Staphylococcus haemolyticus from a laboratory-breeding mouse

ObjectiveTo analysis and identify a bacterium strain isolated from laboratory breeding mouse far away from a hospital.MethodsPhenotype of the isolate was investigated by conventional microbiological methods, including Gram–staining, colony morphology, tests for haemolysis, catalase, coagulase, and antimicrobial susceptibility test. The mecA and 16S rRNA genes were amplified by the polymerase chain reaction (PCR) and sequenced. The base sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by phylogenetic analysis and multiple sequence alignment.ResultsThe isolate in this study was a gram positive, coagulase negative, and catalase positive coccus. The isolate was resistant to oxacillin, methicillin, penicillin, ampicillin, cefazolin, ciprofloxacin erythromycin, et al. PCR results indicated that the isolate was mecA gene positive and its 16S rRNA was 1 465 bp. Phylogenetic analysis of the resultant 16S rRNA indicated the isolate belonged to genus Saphylococcus, and multiple sequence alignment showed that the isolate was Saphylococcus haemolyticus with only one base difference from the corresponding 16S rRNA deposited in the GenBank.Conclusions16S rRNA gene sequencing is a suitable technique for non–specialist researchers. Laboratory animals are possible sources of lethal pathogens, and researchers must adapt protective measures when they manipulate animals.     

Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of 23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.     

Synthesis of a large precursor to ribosomal RNA in a mutant of Escherichia coli

A mutant of E. coli, isolated by Kindler and Hofschneider as a strain defective in RNase III activity, forms a 30S precursor of ribosomal RNA ("30S pre-rRNA"). The half-life of the 30S pre-rRNA in growing cells at 30 degrees , estimated by the rate of specific (3)[H]uridine incorporation, is about 1 min. In rifampicin-treated cells, the RNA is metabolized to mature rRNA with a half-life of about 2 min.The 30S pre-rRNA has been highly purified. DNA-RNA hybridization tests demonstrate that it contains both 16S and 23S rRNA sequences. Also, in cultures treated with rifampicin, the cleavage products of radioactive 30S pre-rRNA include 25S and 17.5S RNA species, destined to becomes 23S and 16S rRNA. Thus, each 30S chain probably contains one 16S and one 23S RNA sequence, as well as additional sequences. Two independent techniques indicate that the additional portions account for about 27% of the total lenght: (1) By comparison to the sedimentation rate and electrophoretic mobility of marker RNAs, the 30S pre-RNA has an apparent molecular weight of 2.3 x 10(6) +/- 5%, or 28% more than the sum of 16S and 23S rRNA; (2) 27% of the 30S pre-rRNA is not competed away from hybridization by mature 16S and 23S rRNA.Thus, bacteria appear to make a pre-rRNA similar in some respects to that observed in eukaryotes; though in normal E. coli cells, the pre-rRNA is ordinarily cleaved endonucleolytically during its formation.     

Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity

Ribosomal protein S5 is critical for small ribosomal subunit (SSU) assembly and is indispensable for SSU function. Previously, we identified a point mutation in S5, (G28D) that alters both SSU formation and translational fidelity in vivo, which is unprecedented for other characterized S5 mutations. Surprisingly, additional copies of an extraribosomal assembly factor, RimJ, rescued all the phenotypes associated with S5(G28D), including fidelity defects, suggesting that the effect of RimJ on rescuing the miscoding of S5(G28D) is indirect. To understand the underlying mechanism, we focused on the biogenesis cascade and observed defects in processing of precursor 16S (p16S) rRNA in the S5(G28D) strain, which were rescued by RimJ. Analyses of p16S rRNA-containing ribosomes from other strains further supported a correspondence between the extent of 5^′ end maturation of 16S rRNA and translational miscoding. Chemical probing of mutant ribosomes with additional leader sequences at the 5^′ end of 16S rRNA compared to WT ribosomes revealed structural differences in the region of helix 1. Thus, the presence of additional nucleotides at the 5^′ end of 16S rRNA could alter fidelity by changing the architecture of 16S rRNA in translating ribosomes and suggests that fidelity is governed by accuracy and completeness of the SSU biogenesis cascade.     

Synovitis of the wrist caused by Mycobacterium florentinum

Mycobacterium florentinum is a newly identified, rare, slow-growing species of nontuberculous mycobacteria (NTM). Here, we report a case of M. florentinum-induced synovitis of the wrist in an immunocompromised Japanese patient. M. florentinum was identified by sequence analysis of the rpoB, hsp65, and 16S rRNA genes. The M. florentinum strain in this study could not be differentiated from certain M. triplex strains by the hsp65 or 16S rRNA sequences alone, because they occasionally shared more than 99 % sequence identity. The isolated M. florentinum strain was only susceptible to clarithromycin and amikacin. Initially, the patient was treated with clarithromycin, levofloxacin, and ethambutol, and then with clarithromycin, levofloxacin, and rifampicin. To our knowledge, M. florentinum-induced synovitis has not been previously reported. Our results suggest that, in addition to other well-known pathogenic NTM, the recently identified M. florentinum strain should be considered as a possible cause of synovitis. Moreover, we should be cautious when identifying M. florentinum because this strain closely resembles M. triplex in genotype.     

Prevalence of phenotypic resistance of Staphylococcus aureus isolates to macrolide,lincosamide, streptogramin B,ketolid and linezolid antibiotics in Turkey

The incidence of drug-resistant pathogens differs greatly between countries according to differences in the usage of antibiotics. The purpose of this study was to investigate the phenotypic resistance of 321 methicillin resistance Staphylococcus aureus (MRSA) and 195 methicillin susceptible S. aureus (MSSA) in a total of 516 S. aureus strains to macrolide, lincosamide, streptogramin B (MLS_B), ketolid, and linezolid. Disk diffusion method was applied to determine MLS_B phenotype and susceptibility to different antibiotic agents. It was found that 54.6% of the isolates were resistant to erythromycin (ERSA), 48% to clindamycin, 55% to azithromycin, 58.7% to spiramycin, 34.7% to telithromycin, and 0.4% to quinupristin-dalfopristin, respectively. No strain resistant to linezolid was found. The prevalence of constitutive (cMLS_B), inducible (IMLS_B), and macrolides and type B streptogramins (M/MS_B) among ERSA isolates (237 MRSA, 45 MSSA) was 69.6%, 18.2%, and 12.2% in MRSA and 28.9%, 40%, and 31.1% in MSSA, respectively. In conclusions, the prevalence of cMLS_B was predominant in MRSA; while in MSSA strains, iMLS_B and M/MS_B phenotype were more higher than cMLS_B phenotype resistance. The resistance to quinupristindalfopristin was very low, and linezolid was considered as the most effective antibiotic against all S.aureus strains.     

Ribosomal resistance: Emerging problems and potential solutions

Many systemic antibiotics use ribosomal inhibition to suppress the replication of bacteria. Current research suggests that resistance to macrolide, lincosamide, and streptogramin B (MLS_B) antibiotics is emerging among clinical isolates of Streptococcus pyogenes and Streptococcus pneumoniae. Erythromycin methylases, encoded by erm genes, modify an essential adenine residue in 23S rRNA and confer cross-resistance to MLS_B antibiotics. More recently, macrolide efflux (mef) genes were identified in isolates of S. pyogenes and S. pneumoniae that show resistance to 14- and 15-membered macrolides (M phenotype). Resistance to MLSB has been associated with the increased use of erythromycin, and the recent emergence of the M phenotype has coincided with the marketing of newer macrolides. However, despite increasing macrolide resistance among clinical isolates of S. pneumoniae, convincing data on treatment failures directly attributable to MLSB or M phenotypes are limited. Possible solutions to emerging MLS_B and M phenotype resistance include the introduction of alternative antibiotics, the more prudent use of antibiotics, combination therapy, molecular diagnostics, enhanced understanding of pharmacodynamic variables, and redefined resistance breakpoints.     

T7 early RNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease 3

The early region of T7 DNA is transcribed as a single unit in a Ribonuclease III-deficient E. coli strain to produce large molecules essentially identical to those produced in vitro by E. coli RNA polymerase. As with the in vitro RNAs, these molecules are cut by purified RNase III in vitro to produce the messenger RNAs normally observed in vivo. Thus, the normal pathway for producing the T7 early messenger RNAs in vivo appears to involve endonucleolytic cleavage by RNase III. The uninfected RNase III-deficient strain contains several RNAs not observed in the parent strain. Patterns of labeling in vivo suggest that the largest of these RNAs, about 1.8 x 10(6) daltons, may be a precursor to the 16S and 23S ribosomal RNAs. When this large molecule is treated in vitro with purified RNase III, molecules the size of precursor 16S and 23S ribosomal RNAs are released; hybridization competition experiments also indicate that the 1.8 x 10(6) dalton RNA does indeed represent ribosomal RNA. Thus, RNase III cleavage seems to be part of the normal pathway for producing at least the 16S and 23S ribosomal RNAs in vivo. Several smaller molecules are also released from the 1.8 x 10(6) dalton RNA by RNase III, but it is not yet established whether any of these contain 5S RNA sequences.     

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.     

Photo-Affinity Labeling of tRNA Binding Sites in Macromolecules. I. Linking of the Phenacyl-p-azide of 4-Thiouridine in (Escherichia coli) Valyl-tRNA to 16S RNA at the Ribosomal P Site

The phenacyl-p-azide of 4-thiouridine in (E. coli) tRNA₁^Val was prepared for use as a photo-affinity probe of tRNA binding sites on ribosomes. The derivatized tRNA was 90-100% as active as control tRNA for aminoacylation, nonenzymatic binding to the ribosomal P site, elongation factor Tu(EFTu)-dependent binding to the A site, EFTu-GTP-aa-tRNA ternary complex formation, and transfer of valine into polypeptide. Irradiation of p-azidophenacyl-[³H]valyl-tRNA bound noncovalently to the ribosomal P site resulted in covalent attachment of 15-20% of the noncovalently bound tRNA to the ribosomes. The linking occurred exclusively to the 16S RNA of the 30S ribosomal subunit, thus suggesting that the region of the ribosome within 9 Å of the 4-thiouridine of tRNA, when it is bound in the P site, is solely 16S RNA.     

PCR offers no advantage over culture for microbiologic diagnosis in cellulitis

Purpose

Most cases of cellulitis are traditionally attributed to ??-hemolytic Streptococcus and Staphylococcus species, although in most cases, no organism is identified. Development of PCR using the conserved bacterial 16?S rRNA DNA permits identification of bacteria independent of conventional culture approaches and prior use of antibiotics.

Methods

We used PCR-based techniques to identify cellulitis etiology using aspirate samples from affected skin. Saline was infiltrated and aspirated at the site of greatest erythema or at the cellulitic border. Samples were tested for 16?S rRNA DNA, and organism-specific probes used to identify bacteria commonly seen in skin infections.

Results

Aspirates from 32 patients were studied, and 16?S rRNA DNA was detected in nine of these patient samples (28.1?%). Bacterial species were identified by PCR methods in six of these nine samples (66.6?%), with S. aureus and methicillin-resistant S. aureus (MRSA) identified in four and two, respectively, of these samples. Of the patients with positive aspirate bacterial cultures (3/9, 33.3?%), S. aureus and coagulase-negative Staphylococcus (CoNS) were present on cultures of two of the three (both 66.6?%) positive samples. Only in one of the three positive bacterial cultures did the PCR method detect the same organism as was detected by culture. Among patients with positive provider-collected clinical cultures, MRSA was the predominant organism (11/18, 61.1?%) and when present, it was found as the sole organism. Where S. aureus or Streptococcus species were detected by molecular methods, clinical cultures yielded a positive result as well.

Conclusions

PCR-based techniques do not appear to be more sensitive than aspirate cultures for the detection of pathogens in cellulitis.     

Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYO^Sa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYO^Sa. Because of the mode of action of PYO^Sa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYO^Sa; none were observed in the 13 clinical isolates tested. PYO^Sa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYO^Sa and antibiotics raise issues that must be addressed before PYO^Sa is employed clinically. Despite the maintenance of the phage, PYO^Sa does not clear populations of S. aureus. Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise–resurrection dynamics of PYO^Sa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYO^Sa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.

Driven by well-warranted concerns about the increasing frequencies of infections with antibiotic-resistant pathogens, there has been a resurrection of interest in, research on, and clinical trials with a therapy that predates antibiotics by more than 15 y: bacteriophage (1–4). One direction phage therapy research has taken is to engineer lytic (virulent) phages with properties that are anticipated to maximize their efficacy for treating bacterial infections in mammals (5–8). Primary among these properties are 1) a broad host range for the target bacterial species; 2) mechanisms that prevent the generation of envelope or other kinds of high-fitness resistance in the target bacteria (9); 3) the capacity to thwart the innate and adaptive immune systems of bacteria, respectively, restriction-modification and CRISPR-Cas (7, 10, 11); 4) the ability to survive, kill, and replicate on pathogenic bacteria colonizing or infecting mammalian hosts (12, 13); and 5) little or no negative effects on the treated host (9).To these five desired properties for therapeutic bacteriophages, there is a sixth that should be considered: the joint action of these phages and antibiotics (8, 14–17). Phages-only treatment may be reasonable for compassionate therapy, where the bacteria responsible for the infection are resistant to all available antibiotics (18–20). From a practical perspective, however, for phages to become widely employed for treating bacterial infections, they would have to be effective in combination with antibiotics. It would be unethical and unacceptable to clinicians and regulatory agencies to use phage-only therapy for infections that can be effectively treated with existing antibiotics.Although not specifically engineered for these properties, there is a Staphylococcal phage isolated from a therapeutic phage collection from the Eliava Institute in Tbilisi, Georgia, that we call PYO^Sa that on first consideration appears to have all six of the properties required to be an effective agent for therapy. 1) PYO^Sa is likely to have a broad host range for S. aureus. The receptor of this K-like Myoviridae is N-acetylglucosamine in the wall-teichoic acid backbone of Staphylococcus aureus and is shared among most (21), if not all, S. aureus, thereby suggesting PYO^Sa should be able to adsorb to and potentially replicate on and kill a vast number of clinical isolates of S. aureus. 2) S. aureus does not generate classical, surface modification mutants resistant to PYO^Sa. Since the structure of the receptor of PYO^Sa is critical to the viability, replication, and virulence of these bacteria, the modifications in this receptor (22) may not be consistent with the viability or pathogenicity of S. aureus (23). 3) The replication of PYO^Sa is unlikely to be prevented by restriction-modification (RM) or CRISPR-Cas. Despite a genome size of 127 KB, the PYO^Sa phage has no GATC nucleotide restriction sites for the S. aureus restriction enzyme Sau3A1 and only one restriction site, GGNCC (guanine, guanine, any nucleotide, cytosine, cytosine), for the Sau961 restriction endonuclease (24, 25). There is no evidence for a functional CRISPR-Cas system in S. aureus or, to our knowledge, other mechanisms by which S. aureus may prevent the replication of this phage (26). 4) There is evidence that PYO^Sa-like phages can replicate in mammals. Early treatment with a phage with a different name but the same properties as PYO^Sa, Statu^v, prevented mortality in otherwise lethal peritoneal infections of S. aureus in mice (27). A PYO^Sa-like phage has also been successfully used therapeutically in humans (28). 5) No deleterious effects of a PYO^Sa-like phage were observed in recent placebo-controlled trials with volunteers asymptotically colonized by S. aureus (24). 6) Finally, there is evidence to suggest synergy with antibiotics. In vitro, PYO^Sa increased the efficacy of low concentrations of antibiotics for the treatment of biofilm populations of S. aureus (14).With in vitro population and evolutionary dynamic experiments with PYO^Sa and S. aureus Newman in combination with three different bacteriostatic and six different bactericidal antibiotics, we explore just how well PYO^Sa fits the above criteria for combination antibiotic and phage therapy. Our population dynamic experiments indicate that as consequence of the ascent of potentially pathogenic PYO^Sa resistant small colony variants (SCVs), by itself, PYO^Sa does not clear S. aureus infections. After an initial decline in the density of these bacteria when confronted with PYO^Sa, despite the continued presence of these phage, the densities of the bacterial populations return to levels similar to those observed in their absence. Using mathematical models, we present a hypothesis to account for these demise resurrection population dynamics, and the continued maintenance of the phage following the ascent of PYO^Sa resistant SCVs. We test and provide evidence in support of that hypothesis with a DNA sequence analysis of the genetic basis of the SCVs. By combining PYO^Sa with antibiotics, the density of the S. aureus population can be markedly reduced. There are, however, significant differences in the effectiveness of this combination therapy depending on whether the antibiotics and phage are used simultaneously or sequentially and on whether the antibiotics are bacteriostatic or bactericidal. Treatment with PYO^Sa, followed by the administration of bactericidal antibiotics, is more effective in reducing density of these bacterial population than treatment with these antibiotics alone. The methods developed here to evaluate the clinical potential of PYO^Sa in combination with antibiotics and design protocols for treating S. aureus infections with these phages and antibiotics can be employed for these purposes for any phage and bacterium that can be cultured in vitro.     

The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA

An oligoribonucleotide (a 27-mer) that mimics the sarcin/ricin (S/R) domain of Escherichia coli 23S rRNA binds elongation factor EF-G; the K_d is 6.9 μM, whereas for binding to ribosomes it is 0.7 μM. Binding saturates when EF-G and the S/R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G. Binding of EF-G to S/R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP. The effects of mutations of the S/R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides. EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S/R and thiostrepton oligoribonucleotides.