Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and ¹⁵N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with ¹⁵N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.     

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E. coli, Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show that E. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli RRF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with RRF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains between mycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with RRF are located in domains IV and V, those required to specify its interaction with the ribosome are located throughout the molecule.     

Synthesis and Evaluation of Chloramphenicol Homodimers: Molecular Target,Antimicrobial Activity,and Toxicity against Human Cells

As fight against antibiotic resistance must be strengthened, improving old drugs that have fallen in reduced clinical use because of toxic side effects and/or frequently reported resistance, like chloramphenicol (CAM), is of special interest. Chloramphenicol (CAM), a prototypical wide-spectrum antibiotic has been shown to obstruct protein synthesis via binding to the bacterial ribosome. In this study we sought to identify features intensifying the bacteriostatic action of CAM. Accordingly, we synthesized a series of CAM-dimers with various linker lengths and functionalities and compared their efficiency in inhibiting peptide-bond formation in an Escherichia coli cell-free system. Several CAM-dimers exhibited higher activity, when compared to CAM. The most potent of them, compound 5, containing two CAM bases conjugated via a dicarboxyl aromatic linker of six successive carbon-bonds, was found to simultaneously bind both the ribosomal catalytic center and the exit-tunnel, thus revealing a second, kinetically cryptic binding site for CAM. Compared to CAM, compound 5 exhibited comparable antibacterial activity against MRSA or wild-type strains of Staphylococcus aureus, Enterococcus faecium and E. coli, but intriguingly superior activity against some CAM-resistant E. coli and Pseudomonas aeruginosa strains. Furthermore, it was almost twice as active in inhibiting the growth of T-leukemic cells, without affecting the viability of normal human lymphocytes. The observed effects were rationalized by footprinting tests, crosslinking analysis, and MD-simulations.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg²⁺ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.     

Ribosome protection by ABC‐F proteins—Molecular mechanism and potential drug design

Members of the ATP‐binding cassette F (ABC‐F) proteins confer resistance to several classes of clinically important antibiotics through ribosome protection. Recent structures of two ABC‐F proteins, Pseudomonas aeruginosa MsrE and Bacillus subtilis VmlR bound to ribosome have shed light onto the ribosome protection mechanism whereby drug resistance is mediated by the antibiotic resistance domain (ARD) connecting the two ATP binding domains. ARD of the E site bound MsrE and VmlR extends toward the drug binding region within the peptidyl transferase center (PTC) and leads to conformational changes in the P site tRNA acceptor stem, the PTC, and the drug binding site causing the release of corresponding drugs. The structural similarities and differences of the MsrE and VmlR structures likely highlight an universal ribosome protection mechanism employed by antibiotic resistance (ARE) ABC‐F proteins. The variable ARD domains enable this family of proteins to adapt the protection mechanism for several classes of ribosome‐targeting drugs. ARE ABC‐F genes have been found in numerous pathogen genomes and multi‐drug resistance conferring plasmids. Collectively they mediate resistance to a broader range of antimicrobial agents than any other group of resistance proteins and play a major role in clinically significant drug resistance in pathogenic bacteria. Here, we review the recent structural and biochemical findings on these emerging resistance proteins, offering an update of the molecular basis and implications for overcoming ABC‐F conferred drug resistance.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.     

Interplay of the Bacterial Ribosomal A-Site,S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

The conformational properties of the aminoacyl-tRNA binding site (A-site), and its surroundings in the Escherichia coli 30S ribosomal subunit, are of great relevance in designing antibacterial agents. The 30S subunit A-site is near ribosomal protein S12, which neighbors helices h27 and H69; this latter helix, of the 50S subunit, is a functionally important component of an intersubunit bridge. Experimental work has shown that specific point mutations in S12 (K42A, R53A) yield hyper-accurate ribosomes, which in turn confers resistance to the antibiotic ‘paromomycin’ (even when this aminoglycoside is bound to the A-site). Suspecting that these effects can be elucidated in terms of the local atomic interactions and detailed dynamics of this region of the bacterial ribosome, we have used molecular dynamics simulations to explore the motion of a fragment of the E. coli ribosome, including the A-site. We found that the ribosomal regions surrounding the A-site modify the conformational space of the flexible A-site adenines 1492/93. Specifically, we found that A-site mobility is affected by stacking interactions between adenines A1493 and A1913, and by contacts between A1492 and a flexible side-chain (K43) from the S12 protein. In addition, our simulations reveal possible indirect pathways by which the R53A and K42A mutations in S12 are coupled to the dynamical properties of the A-site. Our work extends what is known about the atomistic dynamics of the A-site, and suggests possible links between the biological effects of hyper-accurate mutations in the S12 protein and conformational properties of the ribosome; the implications for S12 dynamics help elucidate how the miscoding effects of paromomycin may be evaded in antibiotic-resistant mutants of the bacterial ribosome.     

Diverse effects of residues 74–78 in ribosomal protein S12 on decoding and antibiotic sensitivity

Ribosomal protein S12 plays key roles in the ribosome’s response to the error-promoting antibiotic streptomycin and in modulating the accuracy of translation. The discovery that substitutions at His76 in S12, distant from the streptomycin binding site, conferred streptomycin resistance in the thermophilic bacterium Thermus thermophilus prompted us to make similar alterations in the S12 protein of Escherichia coli. While, none of the E. coli S12 mutations confers streptomycin resistance, they all have distinct effects on the accuracy of translation. In addition, a subset of the S12 alterations renders the cells hypersensitive to fusidic acid, an inhibitor of the translocation step of translation. These results indicate that the His 76 region of ribosomal protein S12 plays key roles in tRNA selection and translocation steps of protein synthesis, consistent with its interaction with elongation factors EF-Tu and EF-G, as deduced from structural studies of ribosomal complexes.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Escherichia coli persister cells suppress translation by selectively disassembling and degrading their ribosomes

Bacterial persisters are rare, phenotypically distinct cells that survive exposure to multiple antibiotics. Previous studies indicated that formation and maintenance of the persister phenotype are regulated by suppressing translation. To examine the mechanism of this translational suppression, we developed novel methodology to rapidly purify ribosome complexes from persister cells. We purified His‐tagged ribosomes from Escherichia coli cells that over‐expressed HipA protein, which induces persister formation, and were treated with ampicillin to remove antibiotic‐sensitive cells. We profiled ribosome complexes and analyzed the ribosomal RNA and protein components from these persister cells. Our results show that (i) ribosomes in persisters exist largely as inactive ribosomal subunits, (ii) rRNAs and tRNAs are mostly degraded and (iii) a small fraction of the ribosomes remain mostly intact, except for reduced amounts of seven ribosomal proteins. Our findings explain the basis for translational suppression in persisters and suggest how persisters survive exposure to multiple antibiotics.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

Transcriptome-wide identification and expression analyses of ABC transporters in dwarf polish wheat under metal stresses

ABC transporters, which comprise one of the largest protein families, are involved in maintaining osmotic homeostasis, nutrient uptake, pathogen resistance, and metal tolerance. In this study, 30 ABC genes in dwarf polish wheat were characterized and classified into seven subfamilies (ABCA - ABCG). Among them, 24 ABC transporters were newly found in wheat. The expressions of 13 ABC genes in roots and leaves under six metal stresses were also analyzed. All these genes were differentially regulated by Cd (except ABCE2, ABCF4, and ABCF6 in roots), suggesting that these genes participate in Cd transport, sequestration, or uptake. These genes were also differentially regulated by other metals including Cu, Mg, Zn, Fe, and Ni. Results suggest that the expressions of ABC transporters in dwarf polish wheat played important roles in metal transport and detoxification.     

Secondary structure and NMR resonance assignments of the C-terminal DNA-binding domain of Uup protein

ATP-binding cassette (ABC) systems belong to a large superfamily of proteins that couple the energy released from ATP hydrolysis to a wide variety of cellular processes, including not only transport of various molecules, but also gene regulation, and DNA repair. Mutations in the bacterial uup gene, which encodes a cytosolic ABC ATPase, lead to an increase in the frequency of precise excision of transposons Tn10 and Tn5, suggesting a role of the Uup protein in DNA metabolism. Uup is a 72?kDa polypeptide which comprises two ABC domains, separated by a 75-residue linker, and a C-terminal domain (CTD) of unknown function. The Uup protein from Escherichia coli has been shown to bind DNA in vitro, and the CTD domain contributes to the DNA-binding affinity. We have produced and purified uniformly labeled ¹⁵N- and ¹⁵N/¹³C Uup CTD domain (region 528?C635), and assigned backbone and side-chains resonances using heteronuclear NMR spectroscopy. Secondary structure evaluation based on backbone chemical shifts is consistent with the presence of three ??-helices, including two long ones (residues 564?C590 and 601?C632), suggesting that Uup CTD may fold as an intramolecular coiled coil motif. This work provides the starting point towards determining the first atomic structure of a non-ATPase domain within the vast REG subfamily of ABC soluble ATPases.     

Binding of [14C]tuberactinomycin O, an antibiotic closely related to viomycin, to the bacterial ribosome.

The binding of [¹⁴C]tuberactinomycin O, an antibiotic closely related to viomycin, to $\text{E. coli}$ ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA_F (initiation) and inhibition of translocation of peptidyl-tRNA.     

Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii

Summary Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.