NMR Characterization of the Influence of Zinc(II) Ions on the Structural and Dynamic Behavior of the New Delhi Metallo-β-Lactamase-1 and on the Binding with Flavonols as Inhibitors.

New Delhi metallo-β-lactamase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to all common β-lactam antibiotics. Understanding the molecular basis of β-lactam hydrolysis by NDM is crucial for designing NDM inhibitors or β-lactams resistant to their hydrolysis. In this study, for the first time, NMR was used to study the influence of Zn(II) ions on the dynamic behavior of NDM-1.

Ribosomal protein eL42 contributes to the catalytic activity of the yeast ribosome at the elongation step of translation.

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function.

RpbL12 Assists Catalysis by Correctly Positioning the Incoming Aminoacyl-tRNA in the A-Site of 70S Ribosomes.

Introduction: We have recently demonstrated that Lys-65 of the 62GANK65 motif of RpbL12 was affinity labeled with a tRNA analogue, resulting in the loss of activity.

Materials And Methods: In this report, we show that mutations operated at the position of Lys-65 led to an impairment in the activity of the mutant ribosomes, except the K65R or K65H bL12 mutants, suggesting that the only requirement of the reaction catalyzed or facilitated by RpbL12is the positive charge of the side chain of Lys-65. We also demonstrate that Lys-65 did not play any role in the peptidyl transferase reaction with respect to puromycin, but rather assisted the binding of the incoming aminoacyl-tRNA to the ribosomal A-site.

Affinity labelling in situ of the bL12 protein on E. coli 70S ribosomes by means of a tRNA dialdehyde derivative.

In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribosomes; (ii) the stoichiometry of labelling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E. coli bL12 protein is one molecule of PLP covalently bound per polypeptide chain; (vii) the amino acid residue of recombinant bL12 cross-linked with PLP is Lys-65; (viii) Lys-65 of E. coli bL12 corresponds to Lys-53 of eL42 which was previously shown to cross-link with P-site bound tRNAox on human 80S ribosomes in situ; (ix) finally, E.

A Functional Role for the Monomethylated Gln-51 and Lys-53 Residues of the 49GGQTK53 Motif of eL42 from Human 80S Ribosomes.

Background: We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA. We have also recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions.

EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance.

In this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elongator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E.

The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome.

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs.

Resonance assignment of the ribosome binding domain of E. coli ribosomal protein S1.

Ribosomal protein S1 is an essential actor for protein synthesis in Escherichia coli. It is involved in mRNA recruitment by the 30S ribosomal subunit and recognition of the correct start codon during translation initiation. E.

Lys53 of ribosomal protein L36AL and the CCA end of a tRNA at the P/E hybrid site are in close proximity on the human ribosome.

Previously we have shown that the CCA end of a P-tRNA can be crosslinked with the RPL36AL protein of the large subunit of mammalian ribosomes; it belongs to the L44e protein family present in all eukaryotic and archaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for a tRNA at the P/E hybrid site, as a tRNA in all other tRNA positions of pre-translocational ribosomes could not be crosslinked with a ribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E-tRNA, but could also connect the ultimate A of this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues, as well as glutaminyl residue 51), 4) Q51 is part of a conserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl-tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about 50 %, might indicate that protein RPL36AL is a preferential target for regulation.

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome.

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1.

Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway.

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions.