RNase P of the Cyanophora paradoxa cyanelle: a plastid ribozyme.
Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs. Ubiquitous across all three kingdoms of life, the composition and functional contributions of the RNA and protein components of RNase P differ between the kingdoms. RNA-alone catalytic activity has been reported throughout bacteria, but only for some archaea, and only as trace activity for eukarya. Available information for RNase P from photosynthetic organelles points to large differences to bacterial as well as to eukaryotic RNase P: for spinach chloroplasts, protein-alone activity has been discussed; for RNase P from the cyanelle of the glaucophyte Cyanophora paradoxa, a type of organelle sharing properties of both cyanobacteria and chloroplasts, the proportion of protein was found to be around 80% rather than the usual 10% in bacteria. Furthermore, the latter RNase P was previously found catalytically inactive in the absence of protein under a variety of conditions; however, the RNA could be activated by a cyanobacterial protein, but not by the bacterial RNase P protein from Escherichia coli. Here we demonstrate that, under very high enzyme concentrations, the RNase P RNA from the cyanelle of C. paradoxa displays RNA-alone activity well above the detection level. Moreover, the RNA can be complemented to a functional holoenzyme by the E. coli RNase P protein, further supporting its overall bacterial-like architecture. Mutational analysis and domain swaps revealed that this A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, since changes as little as a single point mutation or a base pair identity switch at positions that are not part of the universally conserved catalytic core led to a complete loss of RNA-alone activity. Likely related to this low robustness, extensive structural changes towards an E. coli-type P5-7/P15-17 subdomain as a canonical interaction site for tRNA 3'-CCA termini could not be coaxed into increased ribozyme activity.
Li
Dan
D
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Willkomm
Dagmar K
DK
Schön
Astrid
A
Hartmann
Roland K
RK
D001483:Base Sequence; D000458:Cyanobacteria / Q000201:enzymology*; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D015388:Organelles / Q000201:enzymology; D010957:Plasmids; D018087:Plastids / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D043262:Ribonuclease P / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D020035:Holoenzymes; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D011994:Recombinant Proteins; D012343:RNA, Transfer; D043262:Ribonuclease P; C470865:ribonuclease P, E coli