Role of the D arm and the anticodon arm in tRNA recognition by eubacterial and eukaryotic RNase P enzymes.
Truncated precursor tRNAs lacking the D arm or anticodon arm were studied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm still allowed 5'-processing by E. coli RNase P, but strongly impaired maturation by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activities from all three organisms. Inhibition kinetics and gel retardation studies showed that deletion of the D arm leads to low-affinity binding to E. coli RNase P RNA (M1 RNA). However, the E. coli enzyme appears to form sufficiently strong contacts in the region of the T arm, acceptor stem, and CCA terminus to still allow productive enzyme-substrate interaction even in the absence of the structural contribution provided by the D arm. Pb(2+)-induced hydrolysis of a tRNAGly from T. thermophilus gave identical cleavage patterns in the D arm and anticodon loop in the absence and presence of E. coli M1 RNA, whereas lead hydrolysis was strongly reduced at the CUCCAA 3'-terminus due to the presence of the enzyme.
Hardt
W D
WD
Institut für Biochemie, Freie Universität Berlin, FRG.
Schlegl
J
J
Erdmann
V A
VA
Hartmann
R K
RK
D000926:Anticodon / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002414:Cations, Monovalent; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D007854:Lead / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009694:Nucleic Acid Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012353:RNA, Transfer, Gly / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology
D000926:Anticodon; D002414:Cations, Monovalent; D029968:Escherichia coli Proteins; D009694:Nucleic Acid Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012353:RNA, Transfer, Gly; D007854:Lead; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P; C470865:ribonuclease P, E coli