Interaction of translation initiation factor IF1 with the E. coli ribosomal A site

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.     

Interactions between 23S rRNA and tRNA in the ribosomal E site

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.     

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.     

Point mutations in the 23 S rRNA genes of four lincomycin resistant Nicotiana plumbaginifolia mutants could provide new selectable markers for chloroplast transformation

Summary Experiments designed to establish stable chloroplast transformation require selectable marker genes encoded by the chloroplast genome. The antibiotic lincomycin is a specific inhibitor of chloroplast ribosomal activity and is known to bind to the large ribosomal subunit. We have investigated a defined region of the chloroplast 23 S rRNA genes from four lincomycin resistant Nicotiana plumbaginifolia mutants and from wild-type N. plumbaginifolia. The mutants LR415, LR421 and LR446 have A to G transitions at positions equivalent to the nucleotides 2058 and 2059 in the Escherichia coli 23 S rRNA. The mutant, LR400, possesses a G to A transition at a position corresponding to nucleotide 2032 of the E. coli 23 S rRNA.     

Protected nucleotide G2608 in 23S rRNA confers resistance to oxazolidinones in E. coli

The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum of Gram-positive bacterial pathogens including those resistant to other antibiotics. These drugs specifically inhibit protein biosynthesis whereas DNA and RNA synthesis are not affected. Although biochemical and genetic studies indicate that oxazolidinones target the ribosomal peptidyltransferase center, other investigations suggest that they interact with different regions of ribosomes. Thus, the exact binding site and mechanism of action have remained elusive. Here, we study, by use of base-specific reagents, the effect of the oxazolidinones on the chemical protection footprinting patterns of the 23S rRNA. We report: (i) reproducible protection of G2607 and G2608 of 23S rRNA by a potent oxazolidinone on a ribosome.tRNA.mRNA complex; (ii) no protections were observed on 70S ribosomes devoid of tRNA and mRNA; (iii) EF-G also weakly protected G2607 and G2608; (iv) mutations at G2608 conferred resistance to the oxazolidinones in Escherichia coli cells; and (v) G2607 and G2608 occur near the exit to the peptide tunnel on the 50S subunit. A mechanism for the pleiotropic action of the oxazolidinones is discussed.     

Inhibition of bacterial translation and growth by peptide nucleic acids targeted to domain II of 23S rRNA.

The objective of this work was to study the inhibitory effects of antisense peptide nucleic acids (PNAs) targeted to domain II of 23S rRNA on bacterial translation and growth. In this paper, we report that PNA(G1138) or peptide-PNA(G1138) targeted to domain II of 23S rRNA can inhibit both translation in vitro (in a cell-free translation system) and bacterial growth in vivo. The inhibitory concentration (IC50) and the minimum inhibiting concentration (MIC) are 0.15 and 10 microM, respectively. The inhibition effect of PNA(G1138) in vitro is somewhat lower than that of tetracycline (IC50 = 0.12 microM), but the MIC of peptide-PNA(G1138) against Escherichia coli is significantly higher than that of tetracycline (MIC = 4 microM). Further studies based on similar colony-forming unit (CFU) assays showed that peptide-PNA(G1138) at 10 microM is bactericidal, but the bactericidal effect is less effective than that of tetracycline. Nevertheless, the results demonstrated that the peptide-PNA(G1138) treatment is bactericidal in a dose- and sequence-dependent manner and that the G1138 site of 23S rRNA is a possible sequence target for designing novel PNA-based antibiotics.     

Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence.

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotide sequences of the E. coli and B. brevis 16S rRNA chains, and of RNAse T1 oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzoic acid and C by bisulfite in naked 16S rRNA, and G by kethoxal in active and inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and T1. These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which 46 percent of the nucleotides of 16S rRNA are involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure.     

Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase.

The ErmE methyltransferase from the erythromycin-producing actinomycete Saccharopolyspora erythraea dimethylates the N-6 position of adenine 2058 in domain V of 23S rRNA. This modification confers resistance to erythromycin and to other macrolide, lincosamide, and streptogramin B antibiotics. We investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance to erythromycin and clindamycin. The degree of resistance corresponds to the level of ermE expression. In turn, ermE expression also correlates with the proportion of 23S rRNA molecules that are dimethylated at adenine 2058. The methyltransferase was isolated in an active, concentrated form from E. coli, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been disrupted by removal of magnesium ions. We conclude that the main features that are specifically recognized by the ErmE methyltransferase are displayed within the primary and secondary structures of 23S rRNA domain V.     

Lincomycin,clindamycin and their applications

Lincomycin and clindamycin are lincosamide antibiotics used in clinical practice. Both antibiotics are bacteriostatic and inhibit protein synthesis in sensitive bacteria. They may even be bactericidal at the higher concentrations that can be reached in vivo . Clindamycin is usually more active than lincomycin in the treatment of bacterial infections, in particular those caused by anaerobic species; and it can also be used for the treatment of important protozoal diseases, e.g. malaria, most effectively in combination with primaquine. Resistance to lincomycin and clindamycin may be caused by methylation of 23S ribosomal RNA, modification of the antibiotics by specific enzymes or active efflux from the periplasmic space.     

Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis.

The ribosomal 5S RNA gene from the rrnB operon of E. coli was mutagenised in vitro using a synthetic oligonucleotide hybridised to M13 ssDNA containing that gene. The oligonucleotide corresponded to the 5S RNA sequence positions 34 to 51 and changed the guanosine at position 41 to a cytidine. The DNA containing the desired mutation was identified by dot blot hybridisation and introduced back into the plasmid pKK 3535 which contains the total rrnB operon in pBR 322. Plasmid coded 5S rRNA was selectively labeled with 32p using a modified maxi-cell system, and the replacement of guanosine G41 by cytidine was confirmed by RNA sequencing. The growth of cells containing mutant 5S rRNA was not altered by the base change, and the 5S rRNA was processed and incorporated into 50S ribosomal subunits and 70S ribosomes. The structure of wildtype and mutant 5S rRNA was compared by chemical modification of accessible guanosines with kethoxal and limited enzymatic digestion using RNase T1 and nuclease S1. These results showed that the wildtype and mutant 5S rRNA do not differ significantly in their structure. Furthermore, the formation, interconversion and stability of the two 5S rRNA A- and B-conformers are unchanged.     

Molecular cloning and characterization of two lincomycin-resistance genes, ImrA and ImrB, from Streptomyces lincolnensls 78–11

Two different lincomycin-resistance determinants (lmrA and lmrB) from Streptomyces lincolnensis 78-11 were cloned in Streptomyces lividans 66 TK23. The gene lmrA was localized on a 2.16 kb fragment, the determined nucleotide sequence of which encoded a single open reading frame 1446 bp long. Analysis of the deduced amino acid sequence suggested the presence of 12 membrane-spanning domains and showed significant similarities to the methylenomycin-resistance protein (Mmr) from Streptomyces coelicolor, the QacA protein from Staphylococcus aureus, and several tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria, as well as to some sugar-transport proteins from Escherichia coli. The lmrB gene was actively expressed from a 2.7 kb fragment. An open reading frame of 837 bp could be localized which encoded a protein that was significantly similar to 23S rRNA adenine(2058)-N-methyltransferases conferring macrolide-lincosamide-streptogramin resistance. LmrB also had putative rRNA methyltransferase activity since lincomycin resistance of ribosomes was induced in lmrB-containing strains. Surprisingly, both enzymes, LmrA and LmrB, had a substrate specificity restricted to lincomycin and did not cause resistance to other lincosamides such as celesticetin and clindamycin, or to macrolides.     

Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis

Accessible single-strand bases in Xenopus laevis 28 S ribosomal RNA (rRNA) Domain V, the peptidyl transferase region, were determined by chemical modification with dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulfonate and kethoxal, followed by primer extension. The relative accessibilities of three rRNA substrates were compared: deproteinized 28 S rRNA under non-denaturing conditions (free 28 S rRNA), 60 S subunits and 80 S ribosomes. Overall, our experimental results support the theoretical secondary structure model of Domain V derived by comparative sequence analysis and compensatory base-pair changes, and support some theoretical tertiary interactions previously suggested by covariation. The 60 S subunits and 80 S ribosomes generally show increasing resistance to chemical modification. Bases which are sensitive in free 28 S rRNA but protected in 60 S subunits may be sites for ribosomal protein binding or induced structural rearrangements. Another class of nucleotides is distinguished by its sensitivity in 60 S subunits but protection in 80 S ribosomes; these nucleotides may be involved in subunit-subunit interactions or located at the interface of the ribosome. We found a third class of bases, which is protected in free 28 S rRNA but sensitive in 60 S subunits and/or 80 S ribosomes, suggesting that structural changes occur in Domain V as a result of subunit assembly and ribosome formation. One such region is uniquely hypersensitive in eukaryotic ribosomes but is absent in Escherichia coli ribosomes. Sites that we determined to be accessible on empty 80 S ribosomes could serve as recognition sites for translation components.     

Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA

Transfer RNA protects a characteristic set of bases in 16 S rRNA from chemical probes when it binds to ribosomes. We used several criteria, based on construction of well-characterized in vitro ribosome-tRNA complexes, to assign these proteins to A or P-site binding. All of these approaches lead to similar conclusions. In the A site, tRNA caused protection of G529, G530, A1492 and A1493 (strongly), and A1408 and G1494 (weakly). In the P site, the protected bases are G693, A794, C795, G926 and G1401 (strong), and A532, G966, G1338 and G1339 (weak). In contrast to what is observed for 23 S rRNA, blocking the release of EF-Tu.GDP from the ribosome by kirromycin has no detectable effect on the protection of bases in 16 S rRNA.     

Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase

Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.     

Quantum mechanical studies of lincosamides

Lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens. This group of drugs inhibits the activity of the bacterial ribosome by binding to the 23S RNA of the large ribosomal subunit and blocking protein synthesis. Currently, three X-ray structures of the ribosome in complex with clindamycin are available in the Protein Data Bank, which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin. In this work, we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23S RNA. We studied three lincosamide antibiotics: clindamycin, lincomycin, and pirlimycin at the B3LYP level with the 6-31G** basis set. The focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties. For both functional conformers, we applied natural bond orbital (NBO) analysis and the atoms in molecules (AIM) theory, and calculated the NMR parameters. Based on the results obtained, we were able to show that the structure with the intramolecular hydrogen bond C=O…H-O is the most stable conformer of clindamycin. The charge transfer between the pyrrolidine-derivative ring and the six-atom sugar (methylthiolincosamide), which are linked via an amide bond, was found to be the dominant factor influencing the high stability of this conformer.     

Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated rRNA by ribosomal stalk components and a translocation inhibitor

The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA domains II and VI, and a number of proteins including P0, P1α, P1β, P2α, P2β and L12. Mapping of the rRNA neighborhood of the proteins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components. The absence of protein P0 dramatically increases the sensitivity of the defective ribosome to degradation hampering the RNA footprinting. In ribosomes lacking the P1/P2 complex, protection of a number of nucleotides is detected around positions 840, 880, 1100, 1220–1280 and 1350 in domain II as well as in several positions in the domain VI α-sarcin region. The protection pattern resembles the one reported for the interaction of elongation factors in bacterial systems. The results exclude a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the acidic P proteins. Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2. Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification, thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.     

Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.     

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

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