Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide

In bacteria, ribosome stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here we present a cryo-electron microscopy structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase centre (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural basis for understanding how the drug and the nascent peptide cooperate to inhibit peptide bond formation and induce translation arrest

Molecular mechanism of drug and nascent peptide-dependent ribosome stalling

The ability to monitor the nascent peptide structure and respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such a response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. Examples of bacterial genes regulated by programmed ribosome stalling include secA, tna and ermC. These genes contain an upstream regulatory ORF during the translation of which, ribosome stalling occurs, resulting in upregulation of the downstream gene. For ribosome stalling, the sequence of the nascent peptide encoded by the upstream ORF is critical. In case of ermC, ribosome stalling is also dependent on the presence of a specific antibiotic. The molecular mechanisms of programmed translation arrest are unclear. By using bioinformatics, we carried out a systematic analysis of the upstream regions of inducible macrolide resistance genes and identified various putative regulatory ORFs. The sequences of the peptides encoded in these ORFs were found to contain common sequence motifs, based on which we classified the leader peptides into different groups. Analysis of representative ORFs from each group revealed that programmed ribosome stalling occurs during translation. Investigation of the role of the antibiotic in ribosome stalling revealed that different peptides can cooperate with different antibiotics in order to cause translation arrest. By detailed analysis of ribosome stalling at the regulatory cistron of the ermA resistance gene, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyltransferase center in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the peptidyltransferase center to catalyze peptide bond formation with a subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role

Autism Spectrum Disorders: A Recent Update on Targeting Inflammatory Pathways with Natural Anti-Inflammatory Agents

Autism spectrum disorder (ASD) is a heterogeneous category of developmental psychiatric disorders which is characterized by inadequate social interaction, less communication, and repetitive phenotype behavior. ASD is comorbid with various types of disorders. The reported prevalence is 1% in the United Kingdom, 1.5% in the United States, and ~0.2% in India at present. The natural anti-inflammatory agents on brain development are linked to interaction with many types of inflammatory pathways affected by genetic, epigenetic, and environmental variables. Inflammatory targeting pathways have already been linked to ASD. However, these routes are diluted, and new strategies are being developed in natural anti-inflammatory medicines to treat ASD. This review summarizes the numerous preclinical and clinical studies having potential protective effects and natural anti-inflammatory agents on the developing brain during pregnancy. Inflammation during pregnancy activates the maternal infection that likely leads to the development of neuropsychiatric disorders in the offspring. The inflammatory pathways have been an effective target for the subject of translational research studies on ASD

Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-site of the Peptidyl Transferase Center

The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third from last residue plays a critical role

Growth dependence of <i>inhA</i>/KD/DO on hemin.

<p>Wild type <i>M</i>. <i>smegmatis</i> (WT), <i>inhA</i>/KD/DO and <i>inhA</i>/KD/DO/<i>hemH</i> were grown till mid log phase, washed and dilutions plated on two sets of 7H11 plates, one set supplemented with 50 μg/ml hygromycin, 40 μg/ml hemin and either 0, 10 or 50 μM IPTG, another set supplemented with 50 μg/ml hygromycin and either 0, 10 or 50 μM IPTG. Solid bars (no hemin (0H), shaded bars (40 μg/ml hemin (40H)). This data is representative of 2 independent experiments.</p

IPTG inducible conditional expression vectors with promoter-operator sequences.

<p><b>(A)</b> Vector maps of conditional expression vectors with single lac operator (left) and double lac operator (right); <b>(B)</b> Promoter-operator sequences present in the two conditional expression vectors.</p

Minimum IPTG requirement of the <i>rpoB</i> conditional expression strains.

<p>Cultures of wild-type <i>M</i>. <i>smegmatis</i>, <i>rpoB</i>/KD/SO and <i>rpoB</i>/KD/DO were plated on 7H11 plates supplemented with 50 μg/ml hygromycin and different concentrations of IPTG. Wild-type <i>M</i>. <i>smegmatis</i> mc²155 served as control. The numbers above the agar plates indicate the μM IPTG concentration supplemented in the respective plates.</p