title: Swapping the domains of exoribonucleases RNase II and RNase R: conferring upon RNase II the ability to degrade ds RNA.
abstract: RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.
authors.affiliation: Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.
authors.forename: Rute Gonçalves
authors.initials: RG
authors.lastname: Matos
authors.forename: Ana
authors.initials: A
authors.lastname: Barbas
authors.forename: Paulino
authors.initials: P
authors.lastname: Gómez-Puertas
authors.forename: Cecília Maria
authors.initials: CM
authors.lastname: Arraiano
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D005095:Exoribonucleases; C022535:exoribonuclease II; C080868:ribonuclease R