Structural insight into precursor tRNA processing by yeast ribonuclease P.

Ribonuclease P (RNase P) is a universal ribozyme responsible for processing the 5'-leader of pre-transfer RNA (pre-tRNA). Here, we report the 3.5-angstrom cryo-electron microscopy structures of Saccharomyces cerevisiae RNase P alone and in complex with pre-tRNAPhe The protein components form a hook-shaped architecture that wraps around the RNA and stabilizes RNase P into a "measuring device" with two fixed anchors that recognize the L-shaped pre-tRNA. A universally conserved uridine nucleobase and phosphate backbone in the catalytic center together with the scissile phosphate and the O3' leaving group of pre-tRNA jointly coordinate two catalytic magnesium ions. Binding of pre-tRNA induces a conformational change in the catalytic center that is required for catalysis. Moreover, simulation analysis suggests a two-metal-ion SN2 reaction pathway of pre-tRNA cleavage. These results not only reveal the architecture of yeast RNase P but also provide a molecular basis of how the 5'-leader of pre-tRNA is processed by eukaryotic RNase P.

AI-2 quorum sensing negatively regulates rbf expression and biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is an important pathogen that is capable of forming biofilms on biomaterial surfaces to cause biofilm-associated infections. Autoinducer 2 (AI-2), a universal language for interspecies communication, is involved in a variety of physiological activities, although its exact role in Gram-positive bacteria, especially in S. aureus, is not yet thoroughly characterized. Herein we demonstrate that inactivation of luxS, which encodes AI-2 synthase, resulted in increased biofilm formation and higher polysaccharide intercellular adhesion (PIA) production compared with the wild-type strain in S. aureus NCTC8325. The transcript level of rbf, a positive regulator of biofilm formation, was significantly increased in the luxS mutant. All of the parental phenotypes could be restored by genetic complementation and chemically synthesized 4,5-dihydroxy-2,3-pentanedione, the AI-2 precursor molecule, suggesting that AI-2 has a signaling function to regulate rbf transcription and biofilm formation in S. aureus. Phenotypic analysis revealed that the luxS rbf double mutant produced approximately the same amount of biofilms and PIA as the rbf mutant. In addition, real-time quantitative reverse transcription-PCR analysis showed that the icaA transcript level of the rbf mutant was similar to that of the luxS rbf double mutant. These findings demonstrate that the LuxS/AI-2 system regulates PIA-dependent biofilm formation via repression of rbf expression in S. aureus. Furthermore, we demonstrated that Rbf could bind to the sarX and rbf promoters to upregulate their expression.

Structure of the intact ATM/Tel1 kinase.

The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.