ROLE OF THE D-ARM AND THE ANTICODON ARM IN TRANSFER-RNA RECOGNITION BY EUBACTERIAL AND EUKARYOTIC RNASE P-ENZYMES

Truncated precursor tRNAs lacking the D arm or anticodon arm were stud ied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm sti ll allowed 5'-processing by E. coli RNase P, but strongly impaired mat uration by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activitie s from all three organisms. Inhibition kinetics and gel retardation st udies 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 t o form sufficiently strong contacts in the region of the T arm, accept or stem, and CCA terminus to still allow productive enzyme-substrate i nteraction even in the absence of the structural contribution provided by the D arm. Pb2+-induced hydrolysis of a tRNA(Gly) from T. thermoph ilus 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.