Matrices of Paired Substitutions Show the Effects of tRNA D/T Loop Sequence on Drosophila RNase P and 3′-tRNase Processing

• Published in: The Journal of biological chemistry Vol. 273; no. 2; p. 1015
• Main Authors: Louis Levinger, Rae Bourne, Srinivas Kolla, Edruge Cylin, Kirk Russell, Xudong Wang, Amulya Mohan
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 09-01-1998
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.2.1015

Drosophila RNase P and 3′-tRNase endonucleolytically process the 5′ and 3′ ends of tRNA precursors. We examined the processing kinetics
of normal substrates and the inhibitory effect of the tRNA product on both processing reactions. The product is not a good
RNase P inhibitor, with a K
I approximately 7 times greater than the substrate K
M of ∼200 n m and is a better inhibitor of 3′-tRNase, with a K
I approximately two times the K
M of ∼80 n m .
We generated matrices of substitutions at positions G 18 /U 55 and G 19 /C 56 (two contiguous universally conserved D/T loop base pairs) in Drosophila tRNA His precursors. More than half the variants display a significant reduction in their ability to be processed by RNase P and 3′-tRNase.
Minimal substrates with deleted D and anticodon stems could be processed by RNase P and 3′-tRNase much like full-length substrates,
indicating that D/T loop contacts and D arm/enzyme contacts are not required by either enzyme.
Selected tRNAs that were poor substrates for one or both enzymes were further analyzed using Michaelis-Menten kinetics and
by structure probing. Processing reductions arise principally due to an increase in K
M with relatively little change in V
max , consistent with the remote location of the sequence and structure changes from the processing site for both enzymes. Local
changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.