The synthesis and 16S A-site rRNA recognition of carbohydrate-free aminoglycosides

The first carbohydrate-free aminoglycoside analogs bearing the 2-deoxystreptamine moiety were synthesized from asymmetrically protected 2-deoxystrepamine and subsequently demonstrated to have significant binding to the 16S A-site rRNA target and moderate functional activity.     

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.     

Conserved loop sequence of helix 69 in Escherichia coli 23 S rRNA is involved in A-site tRNA binding and translational fidelity

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

Identification of a structural motif of 23S rRNA interacting with 5S rRNA.

To identify RNA motifs interacting with 5S rRNA, a systematic evolution of ligands by exponential enrichment experiment was applied. Some of the resulting RNA aptamers contained a consensus sequence similar to the sequence in the loop region of helix 89 of 23S rRNA. We show that the synthetic helix 89 RNA motif indeed interacted with 5S rRNA and that the region around loop B of 5S rRNA was involved in this interaction. These results suggest the presence of a novel RNA-RNA interaction between 23S rRNA and 5S rRNA which may play an important role in the ribosome function.     

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 sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit

The sarcin–ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.     

Enhanced binding of aminoglycoside dimers to a "dimerized" A-site 16S rRNA construct

In this work, we investigated the binding of a series of dimeric aminoglycoside molecules to (i) a 27 nt A-site 16S rRNA construct, and (ii) an artificially grafted 46 nt 'dimerized' A-site 16S rRNA construct. It was observed that the dissociation constants of dimeric aminoglycosides to the dimerized A-site 16S rRNA construct can achieve up to approximately 19-fold enhancement compared to the monomeric aminoglycoside molecules.     

Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective.

The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme

DEx^D/_H proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DEx^D/_H protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding and ATPase activity of DbpA with truncated RNAs. When DbpA was bound with AMPPNP, but not ADP, several additional footprints were detected in helix 93 and the single-stranded region 5′ of helix 90, suggesting binding of the helicase domains of DbpA at these sites. These results propose that DbpA can act at multiple sites and hint at the targets of its biological activity on rRNA.     

Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA

Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.     

Identification of a 23S rRNA Gene Mutation in Clarithromycin-Resistant Helicobacter pylori

Background Transition mutations (A-G) at residue 2143, cognate to position 2058 in the Escherichia coli 23S rRNA gene, have been shown to confer resistance to macrolides in Helicobacter pylori. This study reports the finding that transversion mutations (A-C) can occur at 2143 as well.
Materials and Methods. Three clarithromycin-resistant H. pylori isolated from three different patients after treatment with clarithromycin were analyzed for point mutations by cycle sequencing of a 163-bp amplified region surrounding residue 2143 within the conserved loop of the 23S rRNA gene.
Results. Nucelotide sequence comparisons of a 163-bp amplified product revealed that A-C transversion mutations occurred at position 2143. H. pylori isolated from the patients prior to treatment were susceptible to clarithromycin and displayed no polymorphism at 2143.
Conclusion. This is the first report to show that A-C transversion mutations at position 2143 can confer resistance to clarithromycin in H. pylori and further support the role that mutations at position 2143 play in conferring macrolide resistance in H. pylori.     

The Escherichia coli DEAD protein DbpA recognizes a small RNA hairpin in 23S rRNA

The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA-protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3' end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5' or 3' side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.     

Role of the 5.8S rRNA in ribosome translocation.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.     

Ribosome origins: The relative age of 23S rRNA Domains

The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.     

Definition of bases in 23S rRNA essential for ribosomal subunit association

The ribosome is a two-subunit molecular machine, sporting a working cycle that involves coordinated movements of the subunits. Recent structural studies of the 70S ribosome describe a rather large number of intersubunit contacts, some of which are dynamic during translocation. We set out to determine which intersubunit contacts are functionally indispensable for the association of ribosome subunits by using a modification interference approach. Modification of the N-1 position of A715, A1912, or A1918 in Escherichia coli 50S subunits is strongly detrimental to 70S ribosome formation. This result points to 23S rRNA helices 34 and 69, and thus bridges B2a and B4, as essential for ensuring stability of the 70S ribosome.     

Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.     

Mutations in the intersubunit bridge regions of 23 S rRNA

The large and small subunits of the ribosome are joined by a series of bridges that are conserved among mitochondrial, bacterial, and eukaryal ribosomes. In addition to joining the subunits together at the initiation of protein synthesis, a variety of other roles have been proposed for these bridges. These roles include transmission of signals between the functional centers of the two subunits, modulation of tRNA-ribosome and factor-ribosome interactions, and mediation of the relative movement of large and small ribosomal subunits during translocation. The majority of the bridges involve RNA-RNA interactions, and to gain insight into their function, we constructed mutations in the 23 S rRNA regions involved in forming 7 of the 12 intersubunit bridges in the Escherichia coli ribosome. The majority of the mutants were viable in strains expressing mutant rRNA exclusively but had distinct growth phenotypes, particularly at 30 degrees C, and the mutant ribosomes promoted a variety of miscoding errors. Analysis of subunit association activities both in vitro and in vivo indicated that, with the exception of the bridge B5 mutants, at least one mutation at each bridge site affected 70 S ribosome formation. These results confirm the structural data linking bridges with subunit-subunit interactions and, together with the effects on decoding fidelity, indicate that intersubunit bridges function at multiple stages of protein synthesis.