Hybrid E. coli--Mitochondrial ribonuclease P RNAs are catalytically active.
RNase P is a ribonucleoprotein that cleaves tRNA precursors at their 5'-end. Mitochondrion-encoded RNA subunits of mitochondrial RNase P (mtP-RNA) have been identified in jakobid flagellates such as Reclinomonas americana, in the prasinophyte alga Nephroselmis olivacea, and in several ascomycete and zygomycete fungi. While the structures of ascomycete mtP-RNAs are highly reduced, those of jakobids, prasinophytes, and zygomycetes retain most conserved features of their bacterial counterparts. Therefore, these mtP-RNAs might be active in vitro in the absence of a protein subunit, as are bacterial P-RNAs. Here we present a comparative structural analysis including seven newly characterized jakobid mtP-RNAs. We investigate ribozyme activities of mtP-RNAs and find that even the most bacteria-like molecules of jakobids are inactive in vitro. However, when certain domains of jakobid and N. olivacea mtP-RNAs are replaced with those from Escherichia coli, these hybrid RNAs show catalytic activity. In vitro mutagenesis of these hybrid mtP-RNAs shows that various structural elements play a critical role in ribozyme catalysis and provide further support for the presence of these elements in mtP-RNAs. These include GNRA tetraloops in helix P14 and P18 of Jakoba libera, and a remnant P3 pairing in Seculamonas ecuadoriensis. Finally, we will discuss reasons for the failure of mtP-RNAs to show catalytic activity in the absence of P-proteins based on our mutagenesis analysis.
Seif
Elias
E
Canadian Institute for Advanced Research, Robert-Cedergren Centre for Bioinformatics and Genomics, Département de Biochimie, Université de Montréal, Montréal, Québec, H3T 1J4, Canada.
Cadieux
Alexandre
A
Lang
B Franz
BF
D002678:Chimera / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D043262:Ribonuclease P / Q000378:metabolism*; D013045:Species Specificity
D012329:RNA, Bacterial; D016337:RNA, Catalytic; D000077278:RNA, Mitochondrial; D012313:RNA; D043262:Ribonuclease P