Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes.

To investigate the function of base 792 of 16S rRNA in 30S ribosomes of Escherichia coli, the wild-type (adenine) residue was changed to guanine, cytosine, or uracil by oligonucleotide-directed mutagenesis. Each base change conferred a unique phenotype on the cells. Cells containing plasmid pKK3535 with G792 or T792 showed no difference in generation time in LB broth containing ampicillin, whereas cells with C792 exhibited a 20% increase in generation time in this medium. To study the effect on cell growth of a homogeneous population of mutant ribosomes, the mutations were cloned into the 16S rRNA gene on pKK3535 carrying a spectinomycin-resistance marker (thymine at position 1192), and the cells were grown with spectinomycin. Cells containing G792 or C792 showed 16% and 56% increases in generation time, respectively, and a concomitant decrease in 35S assimilation into proteins. Cells with T792 did not grow in spectinomycin-containing medium. Maxicell analyses indicated decreasing ability to form 70S ribosomes from 30S subunits containing guanine, cytosine, or uracil at position 792 in 16S rRNA. It appeared that C792-containing 30S ribosomes had lost the ability to bind initiation factor 3.     

Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA.

A putative base-pairing interaction that determines the folding of the central region of 23S rRNA has been investigated by mutagenesis. Each of the possible base substitutions has been made at the phylogenetically covariant positions adenine-1262 (A1262) and U2017 in Escherichia coli 23S rRNA. Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis. All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits to form translationally active ribosomes. The function of 23S rRNA is largely reestablished by introduction of an alternative G1262.C2017 or U1262.A2017 pair, although neither of these supports polysome formation quite as effectively as the wild-type pair. A 23S rRNA with a C1262.G2017 pair is nonfunctional. In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations. The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also indicate that these nucleotides engage in additional interactions within the ribosome and that these interactions determine what base pairs are acceptable there.     

A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid/base catalyzed ribosomal transpeptidation.     

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.     

Reactivation of denatured proteins by 23S ribosomal RNA: role of domain V.

Escherichia coli ribosome, its 50S subunit, or simply the 23S rRNA can reactivate denatured proteins in vitro. Here we show that protein synthesis inhibitors chloramphenicol and erythromycin, which bind to domain V of 23S rRNA of E. coli, can inhibit reactivation of denatured pig muscle lactate dehydrogenase and fungal glucose-6-phosphate dehydrogenase by 23S rRNA completely. Oligodeoxynucleotides complementary to two regions within domain V (which cover sites of chloramphenicol resistant mutations and the putative A site of the incoming aminoacyl tRNA), but not to a region outside of domain V, also can inhibit the activity. Domain V of 23S rRNA, therefore, appears to play a crucial role in reactivation of denatured proteins.     

The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.     

Specific in situ cleavage of 16S ribosomal RNA of Escherichia coli interferes with the function of initiation factor IF-1.

Specific in situ cleavage of 16S rRNA of E. coli has been accomplished by in vitro treatment of 70S ribosomes ("tight couples") with the bacteriocin cloacin DF13. The defective ribosomes, which have fully lost their ability to sustain polypeptide synthesis, are still able to form initiation on complexes with MS2 RNA, but the kinetics are altered. This is apparently due to an improper functioning of initiation factor IF-1, for the defective ribosomal couples respond normally to dissociation by IF-3 but the dissociation is not stimulated by IF-1. The initiation complexes formed with defective ribosomes are fully reactive with puromycin. Their ability to bind alanyl-tRNA is reduced by about 50% at all concentrations of elongation factor Tu studied. Cleavage of the 16S rRNA, not the release of the terminal fragment from the ribosome, causes the block of protein synthesis and the aberrations observed during initiation and elongation.     

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.     

Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.

Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site. Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified. Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530. We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se. In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex. These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation.     

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.     

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3'' end of the acceptor arm: a model of the tRNA-ribosome complex.

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.     

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.     

Oligonucleotide-directed peptide synthesis in a ribosome- and ribozyme-free system

Peptide bond formation by the ribosome requires 23S rRNA and its interaction with the 3'-CCA end of tRNA. To investigate the possible evolutionary development of the peptidyl transfer reaction, we tried to obtain peptide bond formation without the ribosome or rRNA simply by using a piece of tRNA--an aminoacyl-minihelix--mixed with sequence-specific oligonucleotides that contained puromycin. Peptide bond formation was detected by gel electrophoresis, TLC analysis, and mass spectrometry. Peptide synthesis depended on sequence complementarity between the 3'-CCA sequence of the minihelix and the puromycin-bearing oligonucleotide. However, proximity of the reacting species was not by itself sufficient for peptide bond formation. In addition, imidazole as a catalyst was required. Its role may be similar to the recently proposed mechanism, wherein A2451 of 23S rRNA works as a general base. Thus, peptide bond formation can be achieved with a simple, minimized system that captures the essence of an interaction seen in the ribosome.     

5′-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: Features of translational initiation and reinitiation sites

In a sequence of 214 nucleotides at the 5' terminus of the I gene mRNA, which codes for the lactose repressor protein of Escherichia coli, (i) an untranslated leader sequence of 28 residues precedes the repressor coding region; (ii) a GUG initiates synthesis of the wild-type repressor; (iii) GUG and AUG are the functional initiators for the synthesis of restart polypeptides activated by early I gene amber mutations, confirming previous assignments for these residues based on protein sequencing data; and (iv) sequences complementary to 16S ribosomal RNA provide stronger potential mRNA.16S rRNA interaction at the wild-type initiation site than at the restart sites. When I mRNA is used to direct the formation of initiation complexes in vitro, ribosomes bind only to the wild-type initiator region.A striking feature of the I mRNA sequence is the presence of a number of in-phase GUGs that have not been observed to serve as initiation signals in vivo in the nonsense mutant strains examined. The selective use of potential initiator triplets in the I mRNA leads to the following conclusions. First, when presented with several neighboring initiator triplets at the wild-type initiator region, ribosomes select the one preceded by the strongest appropriately positioned complementarity to the 16S 3' end. Second, ribosomes do not restart after termination simply by moving to the next available initiator codon. Third, the formation of stable secondary structures predicted for the untranslated I mRNA beyond chain-terminating nonsense mutations may prevent ribosome access to some potential reinitiation sites.     

Coregulation of processing and translation: mature 5'' termini of Escherichia coli 23S ribosomal RNA form in polysomes.

In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated.     

23S rRNA positions essential for tRNA binding in ribosomal functional sites

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.     

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.     

A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.     

Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system

Genetic, biochemical, and structural data support an essential role for the ribosomal RNA in all steps of the translation process. Although in vivo genetic selection techniques have been used to identify mutations in the rRNAs that result in various miscoding phenotypes and resistance to known ribosome-targeted antibiotics, these are limited because the resulting mutant ribosomes must be only marginally disabled if they are able to support growth of the cell. Furthermore, in vivo, it is not possible to control the environment in precise ways that might allow for the isolation of certain types of rRNA variants. To overcome these limitations, we have developed an in vitro selection system for the isolation of functionally competent ribosomal particles from populations containing variant rRNAs. Here, we describe this system and present an example of its application to the selection of antibiotic resistance mutations. From a pool of 4,096 23S rRNA variants, a double mutant (A2058U/A2062G) was isolated after iteration of the selection process. This mutant was highly resistant to clindamycin in in vitro translation reactions and yet was not viable in Escherichia coli. These data establish that this system has the potential to identify mutations in the rRNA not readily accessed by comparable in vivo systems, thus allowing for more exhaustive ribosomal genetic screens.     

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.