Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Many clinically useful antibiotics interfere with protein synthesis in bacterial pathogens by inhibiting ribosome function. The sites of action of known drugs are limited in number, are composed primarily of ribosomal RNA (rRNA), and coincide with functionally critical centers of the ribosome. Nucleotide alterations within such sites are often deleterious. To identify functional sites and potential sites of antibiotic action in the ribosome, we prepared a random mutant library of rRNA genes and selected dominant mutations in 16S rRNA that interfere with cell growth. Fifty-three 16S rRNA positions were identified whose mutation inhibits protein synthesis. Mutations were ranked according to the severity of the phenotype, and the detrimental effect of several mutations on translation was verified in a specialized ribosome system. Analysis of the polysome profiles of mutants suggests that the majority of the mutations directly interfered with ribosome function, whereas a smaller fraction of mutations affected assembly of the small ribosomal subunit. Twelve of the identified mutations mapped to sites targeted by known antibiotics, confirming that deleterious mutations can be used to identify antibiotic targets. About half of the mutations coincided with known functional sites in the ribosome, whereas the rest of the mutations affected ribosomal sites with less clear functional significance. Four clusters of deleterious mutations in otherwise unremarkable ribosomal sites were identified, suggesting their functional importance and potential as antibiotic targets.     

The phylogenetically conserved doublet tertiary interaction in domain III of the large subunit rRNA is crucial for ribosomal protein binding.

Previous phylogenetic analysis of rRNA sequences for covariant base changes has identified approximately 20 potential tertiary interactions. One of these is present in domain III of the large subunit rRNA and consists of two adjacent Watson-Crick base pairs that, in Saccharomyces cerevisiae 26S rRNA, connect positions 1523 and 1524 to positions 1611 and 1612. This interaction would strongly affect the structure of an evolutionarily highly conserved region that acts as the binding site for the early-assembling ribosomal proteins L25 and EL23 of S. cerevisiae and Escherichia coli, respectively. To assess the functional importance of this tertiary interaction, we determined the ability of synthetically prepared S. cerevisiae ribosomal protein L25 to associate in vitro with synthetic 26S rRNA fragments containing sequence variations at positions 1523 and 1524 and/or positions 1611 and 1612. Mutations that prevent the formation of both base pairs abolished L25 binding completely, whereas the introduction of compensatory mutations fully restored protein binding. Disruption of only the U1524.A1611 pair reduced L25 binding to approximately 30% of the value shown by the wild-type 26S rRNA fragment, whereas disruption of the G1523.C1612 base pair resulted in almost complete loss of protein binding. These results strongly support the existence and functional importance of the proposed doublet tertiary interaction in domain III of the large subunit rRNA.     

High rate of macrolide resistance in Staphylococcus aureus strains from patients with cystic fibrosis reveals high proportions of hypermutable strains

Incidence of resistance to erythromycin at our institution reached 53% in 122 Staphylococcus aureus isolates obtained from patients with cystic fibrosis (CF) from 1997 to 1999. Macrolide-resistance genes were sought for in 20 erythromycin-resistant isolates from 9 patients with CF by use of polymerase chain reaction; 13 strains did not contain any known macrolide-resistance genes. Sequence of ribosomal genes rrl (23S rRNA), rplD (L4 protein), and rplV (L22 protein) revealed the presence of mutations in the target site of macrolides in 15 of the 20 isolates. A higher proportion of hypermutator strains was observed in a group of 89 CF staphylococcal isolates, compared with that in the 74 non-CF control isolates (13/89 vs. 1/74 with resistance to rifampin [P=.0045]; 9/89 vs. 1/74 with resistance to streptomycin [P=.04]). Various mutations or deletions of the mutator mutS gene were found not only in 5 of 11 hypermutable strains but also in 3 nonhypermutable strains harboring a large number of ribosomal mutations. The presence of a high proportion of hypermutable strains might explain the adaptation of certain strains in the patients, as well as the emergence of macrolide resistance as a result of antibiotic selective pressure in CF.     

Translational regulation of the L11 ribosomal protein operon of Escherichia coli: analysis of the mRNA target site using oligonucleotide-directed mutagenesis.

The L11 ribosomal protein operon in Escherichia coli consists of the genes for proteins L11 and L1 and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the translation initiation site of the first L11 cistron. Several mutant plasmid molecules carrying altered nucleotide sequences in the L1 target site were constructed by site-directed in vitro mutagenesis using synthetic oligodeoxyribonucleotides. Specifically, we examined the importance of a presumptive double-stranded stem structure that is common among L1 binding sites on rRNA from a variety of organisms and in L11 mRNA. Mutational alterations that disrupt the stem structure were found to abolish translational regulation as analyzed both in vitro and in vivo. Two of the mutations were combined so that the stem structure was restored but with a different primary nucleotide sequence. This double mutant was shown to restore the original phenotype, the ability to be translationally regulated by L1. These experiments show the importance of the stem structure, but not its primary sequence, for the interaction of L1 with the mRNA and support the concept that mRNA target sites share some structural features with the corresponding ribosomal protein binding sites of rRNA.     

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.     

Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity

Aminoglycoside ototoxicity has been related to a surprisingly large number of cellular structures and metabolic pathways. The finding that patients with mutations in mitochondrial rRNA are hypersusceptible to aminoglycoside-induced hearing loss has indicated a possible role for mitochondrial protein synthesis. To study the molecular interaction of aminoglycosides with eukaryotic ribosomes, we made use of the observation that the drug binding site is a distinct domain defined by the small subunit rRNA, and investigated drug susceptibility of bacterial hybrid ribosomes carrying various alleles of the eukaryotic decoding site. Compared to hybrid ribosomes with the A site of human cytosolic ribosomes, susceptibility of mitochondrial hybrid ribosomes to various aminoglycosides correlated with the relative cochleotoxicity of these drugs. Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis. Our results provide experimental support for aminoglycoside-induced dysfunction of the mitochondrial ribosome. We propose a pathogenic mechanism in which interference of aminoglycosides with mitochondrial protein synthesis exacerbates the drugs' cochlear toxicity, playing a key role in sporadic dose-dependent and genetically inherited, aminoglycoside-induced deafness.     

Dominant lethal mutations in a conserved loop in 16S rRNA.

The 530 stem-loop region in 16S rRNA is among the most phylogenetically conserved structural elements in all rRNAs, yet its role in protein synthesis remains mysterious. G-530 is protected from kethoxal attack when tRNA, or its 15-nucleotide anticodon stem-loop fragment, is bound to the ribosomal A site. Based on presently available evidence, however, this region is believed to be too remote from the decoding site for this protection to be the result of direct contact. In this study, we use a conditional rRNA expression system to demonstrate that plasmid-encoded 16S rRNA genes carrying A, C, and T point mutations at position G-530 confer a dominant lethal phenotype when expressed in Escherichia coli. Analysis of the distribution of plasmid-encoded 16S rRNA in ribosomal particles, following induction of the A-530 mutation, shows that mutant rRNA is present both in 30S subunits and in 70S ribosomes. Little mutant rRNA is found in polyribosomes, however, indicating that the mutant ribosomes are severely impaired at the stage of polysome formation and/or stability. Detailed chemical probing of mutant ribosomal particles reveals no evidence of structural perturbation within the 16S rRNA. Taken together, these results argue for the direct participation of G-530 in ribosomal function and, furthermore, suggest that the dominant lethal phenotype caused by these mutations is due primarily to the mutant ribosomes blocking a crucial step in protein synthesis after translational initiation.     

A functional peptide encoded in the Escherichia coli 23S rRNA.

A pentapeptide open reading frame equipped with a canonical ribosome-binding site is present in the Escherichia coli 23S rRNA. Overexpression of 23S rRNA fragments containing the mini-gene renders cells resistant to the ribosome-inhibiting antibiotic erythromycin. Mutations that change either the initiator or stop codons of the peptide mini-gene result in the loss of erythromycin resistance. Nonsense mutations in the mini-gene also abolish erythromycin resistance, which can be restored in the presence of the suppressor tRNA, thus proving that expression of the rRNA-encoded peptide is essential for the resistance phenotype. The ribosome appears to be the likely target of action of the rRNA-encoded pentapeptide, because in vitro translation of the peptide mini-gene decreases the inhibitory action of erythromycin on cell-free protein synthesis. Thus, the new mechanism of drug resistance reveals that in addition to the structural and functional role of rRNA in the ribosome, it may also have a peptide-coding function.     

Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action

Differences between the structures of bacterial, archaeal, and eukaryotic ribosomes account for the selective action of antibiotics. Even minor variations in the structure of ribosomes of different bacterial species may lead to idiosyncratic, species-specific interactions of the drugs with their targets. Although crystallographic structures of antibiotics bound to the peptidyl transferase center or the exit tunnel of archaeal (Haloarcula marismortui) and bacterial (Deinococcus radiodurans) large ribosomal subunits have been reported, it remains unclear whether the interactions of antibiotics with these ribosomes accurately reflect those with the ribosomes of pathogenic bacteria. Here we report X-ray crystal structures of the Escherichia coli ribosome in complexes with clinically important antibiotics of four major classes, including the macrolide erythromycin, the ketolide telithromycin, the lincosamide clindamycin, and a phenicol, chloramphenicol, at resolutions of ∼3.3 Å–3.4 Å. Binding modes of three of these antibiotics show important variations compared to the previously determined structures. Biochemical and structural evidence also indicates that interactions of telithromycin with the E. coli ribosome more closely resembles drug binding to ribosomes of bacterial pathogens. The present data further argue that the identity of nucleotides 752, 2609, and 2055 of 23S ribosomal RNA explain in part the spectrum and selectivity of antibiotic action.     

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.     

Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome

The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the alpha-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.     

Mapping of Escherichia coli Ribosomal Components Involved in Peptidyl Transferase Activity

The method of affinity labeling has been used to identify protein components of 50S ribosomal subunits involved in peptidyl transferase activity. E. coli 50S ribosomal subunits were mapped by reaction with the N-bromoacetyl analog of chloramphenicol, an antibiotic known to interact specifically with the active center of the enzyme. The synthetic analog competes with chloramphenicol in binding to 50S ribosomal subunits and inhibits peptidyl transferase activity. It attaches covalently to the ribosome under appropriate conditions and causes an irreversible loss in peptidyl transferase activity. The reagent specifically alkylates cysteine residues of proteins L2 and L27.     

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.     

Location of eight ribosomal proteins on the surface of the 50S subunit from Escherichia coli.

Eight ribosomal proteins, L9, L11, L15, L17, L18, L19, L23, and L29, have been localized on the surface of the 50S subunit from Escherichia coli by immunoelectron microscopy. The specificity of the antibody binding site was demonstrated by stringent absorption experiments. For each protein, the antibody attachment site was localized on the two characteristic views of the 50S subunit. Thus, each protein could be located in a confined region on the three-dimensional structural model of the 50S subunit.     

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.     

Crystal structure of RlmAI: implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site

The RlmA class of enzymes (RlmA(I) and RlmA(II)) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Gram-positive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli RlmA(I) at 2.8-A resolution, providing 3D structure information for the RlmA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each RlmA(I) molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric RlmA(I) dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-l-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the RlmA(I) dimer.     

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.     

Interaction of Escherichia coli ribosomal protein S1 with ribosomes.

The binding affinity of Escherichia coli ribosomal protein S1 for 30S ribosomal particles has been determined by a sucrose gradient band sedimentation technique; the association constant (K) for the binding of one S1 protein per active 30S ribosomal subunit is approximately 2 X 10(8) M-1. The involvement of the two polynucleotide binding sites of S1 protein (site I binding single-stranded DNA or RNA, and site II binding single-stranded RNA only) in the S1--ribosomal interaction have been examined by competition experiments with polynucleotides of known affinity for the two sites. We find that site I does not contribute to the interaction; site II binding appears to provide a major part of the binding free energy, presumably by interaction of S1 with the 16S rRNA of the 30S particle. The remaining binding free energy is probably derived from the interaction of S1 protein with other proteins of the 30S subunit. The affinity of S1 for 70S ribosomes is about the same as that for the 30S subunit; the affinity of S1 for 50S subunits is much less. Binding affinities and stoichiometries of S1 protein with "inactive" 30S ribosomal subunits have also been examined.     

Translational Control of Hemoglobin Synthesis by an Initiation Factor Required for Recycling of Ribosomes and for their Binding to Messenger RNA

The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 37 degrees requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes.These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor.