title: Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis.
abstract: We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000-fold when the 5' flank was shortened from 3 to 1 nt. Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt -2 in the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes. We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1. All cleavage sites were at least 2 nt away from the 5' and 3' ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.
authors.affiliation: Institut für Biochemie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Hansen
authors.forename: T
authors.initials: T
authors.lastname: Pfeiffer
authors.forename: T
authors.initials: T
authors.lastname: Zuleeg
authors.forename: S
authors.initials: S
authors.lastname: Limmer
authors.forename: J
authors.initials: J
authors.lastname: Ciesiolka
authors.forename: R
authors.initials: R
authors.lastname: Feltens
authors.forename: R K
authors.initials: RK
authors.lastname: Hartmann
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D020035:Holoenzymes / Q000378:metabolism; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D020035:Holoenzymes; D009841:Oligonucleotides; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli