Abstract
Background:
3′-Terminal CCA-containing tRNAs and precursors are resistant to action of RNase BN/RNase Z.
Results:
Arg
274
and the two C residues are required for protection by the CCA sequence.
Conclusion:
Presence of Arg
274
and CC sequence prevents RNA substrate from moving into the RNase catalytic site.
Significance:
This mechanism explains how mature tRNAs are protected from removal of the CCA sequence by a processing RNase.
In many organisms, 3′ maturation of tRNAs is catalyzed by the endoribonuclease, RNase BN/RNase Z, which cleaves after the discriminator nucleotide to generate a substrate for addition of the universal CCA sequence. However, tRNAs or tRNA precursors that already contain a CCA sequence are not cleaved, thereby avoiding a futile cycle of removal and readdition of these essential residues. We show here that the adjacent C residues of the CCA sequence and an Arg residue within a highly conserved sequence motif in the channel leading to the RNase catalytic site are both required for the protective effect of the CCA sequence. When both of these determinants are present, CCA-containing RNAs in the channel are unable to move into the catalytic site; however, substitution of either of the C residues by A or U or mutation of Arg
274
to Ala allows RNA movement and catalysis to proceed. These data define a novel mechanism for how tRNAs are protected against the promiscuous action of a processing enzyme.
