Pseudomonas aeruginosa regulator PvrA binds simultaneously to multiple pseudo-palindromic sites for efficient transcription activation

Tetracycline repressor(TetR)family regulators(TFRs)are the largest group of DNA-binding transcription factors and are widely distributed in bacteria and archaea.TFRs play vital roles in controlling the expression of various genes and regulating diverse physiological processes.Recently,a TFR protein Pseudomonas virulence regulator A(PvrA),was identified from Pseudomonas aeruginosa as the transcriptional activator of genes involved in fatty acid utilization and bacterial virulence.Here,we show that PvrA can simultaneously bind to multiple pseudo-palindromic sites and upregulate the expression levels of target genes.Cryo-electron microscopy(cryo-EM)analysis indicates the simultaneous DNA recognition mechanism of PvrA and suggests that the bound DNA fragments consist of a distorted B-DNA double helix.The crystal structure and functional analysis of PvrA reveal a hinge region that secures the correct domain motion for recognition of the promiscuous promoter.Additionally,our results showed that mutations disrupting the regulatory hinge region have differential effects on biofilm formation and pyocyanin biosynthesis,resulting in attenuated bacterial virulence.Collectively,these findings will improve the understanding of the relationship between the structure and function of the TetR family and provide new insights into the mechanism of regulation of P.aeruginosa virulence.

Cryo-EM structure of the nucleocapsid-like assembly of respiratory syncytial virus

Respiratory syncytial virus(RSV)is a nonsegmented,negative strand RNA virus that has caused severe lower respiratory tract infections of high mortality rates in infants and the elderly,yet no effective vaccine or antiviral therapy is available.The RSV genome encodes the nucleoprotein(N)that forms helical assembly to encapsulate and protect the RNA genome from degradation,and to serve as a template for transcription and replication.Previous crystal structure revealed a decameric ring architecture of N in complex with the cellular RNA(N-RNA)of 70 nucleotides(70-nt),whereas cryo-ET reconstruction revealed a low-resolution left-handed filament,in which the crystal monomer structure was docked with the helical symmetry applied to simulate a nucleocapsid-like assembly of RSV.However,the molecular details of RSV nucleocapsid assembly remain unknown,which continue to limit our complete understanding of the critical interactions involved in the nucleocapsid and antiviral development that may target this essential process during the viral life cycle.Here we resolve the near-atomic cryo-EM structure of RSV N-RNA that represents roughly one turn of the helical assembly that unveils critical interaction interfaces of RSV nucleocapsid and may facilitate development of RSV antiviral therapy.