M. Clelia Ganoza

Oxazolidinone Antibiotics Target the P Site on Escherichia coli Ribosomes

ABSTRACT The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacteriumhalobium, Staphylococcusaureus, and Escherichiacoli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

Evolutionary Conservation of Reactions in Translation

SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria have shed considerable light on the molecular mechanisms of translation. Structural studies of the protein factors that activate ribosomes also point to many common features in the primary sequence and tertiary structure of these proteins. The reconstitution of the complex apparatus of translation has also revealed new information important to the mechanisms. Surprisingly, the latter approach has uncovered a number of proteins whose sequence and/or structure and function are conserved in all cells, indicating that the mechanisms are indeed conserved. The possible mechanisms of a new initiation factor and two elongation factors are discussed in this context.

Molecular characterization of a prokaryotic translation factor homologous to the eukaryotic initiation factor eIF4A.

Initiation of translation involves a complex series of reactions that result in the formation of an initiation complex at the proper start site of the mRNA. These reactions, particularly those that involve the binding of the mRNA to the small subunit of the ribosome, are not fully understood. Here we show that one of the factors (W2) required to reconstitute translation in E. coli is encoded by the deaD gene which harbors 87% amino acid sequence similarly to the eukaryotic (eIF4A). Antibodies against the eukaryotic eIF4A cross-react with the E. coli protein. We describe the overexpression of the W2 protein from recombinant clones and its purification in one step by the use of a His tag at the N-terminus of its sequence. We report a rapid assay for the W2 protein that scores for initiation and elongation programmed by a native mRNA template. The W2 protein promotes initiation programmed by the mRNA that harbors secondary structures. The W2 protein is not required in standard initiation assays programmed by synthetic mRNAs of defined sequence that lack this feature. We conclude that W2 is an important factor for initiation in eukaryotic and prokaryotic cells.

Post-translational Modification by β-Lysylation Is Required for Activity of Escherichia coli Elongation Factor P (EF-P)

Background: Bacterial elongation factor P (EF-P) was proposed to undergo unique post-translational modification by YjeA and YjeK. Results: We identified β-lysylation in native EF-P and show that β-lysyl-EF-P is the active form. Conclusion: β-Lysylation occurs in EF-P in vivo and is required for activity. Significance: β-Lysylation of EF-P may influence this factor binding to ribosome and regulate bacterial protein synthesis. Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical β-lysyl-lysine. Here we present biochemical evidence for β-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The β-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for β-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing β-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ∼4-fold over the preparations containing unmodified EF-P and/or α-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the β-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.

Interactions of elongation factor EF-P with the Escherichia coli ribosome

EF‐P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthesis. We report that EF‐P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T‐site). EF‐P also protects domain V of the 23S rRNA proximal to the A‐site (50S T‐site) and more strongly the A‐site of 70S ribosomes. We suggest that EF‐P: (a) may play a role in translational fidelity and (b) prevents entry of fMet–tRNA into the A‐site enabling it to bind to the 50S P‐site. We also report that EF‐P promotes a ribosome‐dependent accommodation of fMet–tRNA into the 70S P‐site.

A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.

ABSTRACT We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

Investigating the in vivo activity of the DeaD protein using protein–protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs

Here, we report the use of an in vivo protein–protein interaction detection approach together with focused follow‐up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow‐up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow‐up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large‐scale protein–protein interaction data can be suitable to study protein functions in E. coli. J. Cell. Biochem. 100: 642–652, 2007.

Translation of hepatic mRNA in extracts from wheat germ embryos

Extracts from wheat embryos have been used to study the incorporation of amino acids into TCA insoluble products using hepatic mRNA fractions. The properties of this system are described and compared to the incorporation obtained with poly U and rabbit globin mRNA. SDS-acrylamide gel analysis showed that the major polypeptide synthesized with globin mRNA co-migrates with rabbit globin (15 500 daltons). Rat liver products were numerous, with molecular weights from less than 10000 to greater than 65000 daltons. The KCl concentration for maximum incorporation into TCA precipitable polypeptides with hepatic mRNA was not the optimum KCl concentration for synthesis of complete products.

Ribosome-Dependent ATPase Interacts with Conserved Membrane Protein in Escherichia coli to Modulate Protein Synthesis and Oxidative Phosphorylation

Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

Isolation of a factor that stimulates cleavage of ribosomal bound N-acetyl or N-formyl methionyl tRNAmet f

During the code-dependent condensation of amino acids on ribosomes the amino group of an incoming amino acyl-tRNA attacks the ester linkage at the carboxyl end of the nascent peptide and the 3’ OH group of the ribose of the terminal adenosine of tRNA [ I] . This reaction is catalyzed by peptidyl synthetase, a part of the 50s subunit [2], but the active center of this enzyme has not been isolated free of this particle [3,4]. Instead, indirect approaches with antibiotics that specifically block peptide bond synthesis have been used as probes to study the reaction [l-3]. Other observations suggest that the peptidyl transferase synthesizes ester bonds with a suitable acceptor [6,7], and catalyzes ester hydrolysis if one of the release (R) factors and a cognate nonsense codon [8,9] or a suitable nucleophile [lo] are included. These combined observations suggest that ribosomes have the inherent capacity to hydrolyze ester linkages. We report here that the hydrolytic activity of ribosomes is uncoupled when a high molecular factor from the ribosome-free cytoplasm is added. This reaction is nonsense-codon independent and is blocked by inhibitors of peptide bond formation when the initiator fMet-tRNA is bound to ribosomes.