RESUMEN
The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Precursores del ARN/metabolismo , Ribonucleasas/química , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/ultraestructura , Secuencia de Bases , Microscopía por Crioelectrón , Modelos Moleculares , Precursores del ARN/ultraestructura , Ribonucleasas/ultraestructura , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Especificidad por SustratoRESUMEN
Mini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5ß-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. In vitro binding experiments show that inactive Staphylococcus epidermidis mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.