Pseudouridine Synthase RsuA Confers a Survival Advantage to Bacteria under Streptomycin Stress.

Bacterial ribosome small subunit rRNA (16S rRNA) contains 11 nucleotide modifications scattered throughout all its domains. The 16S rRNA pseudouridylation enzyme, RsuA, which modifies U516, is a survival protein essential for bacterial survival under stress conditions. A comparison of the growth curves of wildtype and RsuA knock-out E. coli strains illustrates that RsuA renders a survival advantage to bacteria under streptomycin stress. The RsuA-dependent growth advantage for bacteria was found to be dependent on its pseudouridylation activity. In addition, the role of RsuA as a trans-acting factor during ribosome biogenesis may also play a role in bacterial growth under streptomycin stress. Furthermore, circular dichroism spectroscopy measurements and RNase footprinting studies have demonstrated that pseudouridine at position 516 influences helix 18 structure, folding, and streptomycin binding. This study exemplifies the importance of bacterial rRNA modification enzymes during environmental stress.

Pseudouridine Synthase RsuA Captures an Assembly Intermediate that Is Stabilized by Ribosomal Protein S17.

The ribosome is a large ribonucleoprotein complex that synthesizes protein in all living organisms. Ribosome biogenesis is a complex process that requires synchronization of various cellular events, including ribosomal RNA (rRNA) transcription, ribosome assembly, and processing and post-transcriptional modification of rRNA. Ribosome biogenesis is fine-tuned with various assembly factors, possibly including nucleotide modification enzymes. Ribosomal small subunit pseudouridine synthase A (RsuA) pseudouridylates U516 of 16S helix 18. Protein RsuA is a multi-domain protein that contains the N-terminal peripheral domain, which is structurally similar to the ribosomal protein S4. Our study shows RsuA preferably binds and pseudouridylates an assembly intermediate that is stabilized by ribosomal protein S17 over the native-like complex. In addition, the N-terminal domain truncated RsuA showed that the presence of the S4-like domain is important for RsuA substrate recognition.

Discovery of a novel small molecular peptide that disrupts helix 34 of bacterial ribosomal RNA.

Despite the advances in modern medicine, antibiotic resistance is a persistent and growing threat to the world. Thus, the discovery and development of novel antibiotics have become crucial to combat multi-drug resistant pathogens. The goal of our research is to discover a small molecular peptide that can disrupt the synthesis of new ribosomes. Using the phage display technique, we have discovered a 7-mer peptide that binds to the second strand of 16S h34 RNA with a dissociation constant in the low micromolar range. Binding of the peptide alters RNA structure and inhibits the binding of the ribosomal RNA small subunit methyltransferase C (RsmC) enzyme that methylates the exocyclic amine of G1207. The addition of this peptide also increases the lag phase of bacterial growth. Introduction of chemical modifications to increase the binding affinity of the peptide to RNA, its uptake and stability can further improve the efficacy of the peptide as an antibiotic agent against pathogenic bacteria.