The T loop structure is dispensable for substrate recognition by tRNase ZL.

tRNA 3'-processing endoribonucleases (tRNase Z, or 3'-tRNase; EC 3.1.26.11) are enzymes that remove 3'-trailers from pre-tRNAs. An about 12-base-pair stem, a T loop-like structure, and a 3'-trailer were considered to be the minimum requirements for recognition by the long form (tRNase ZL) of tRNase Z; tRNase ZL can recognize and cleave a micro-pre-tRNA or a hooker/target RNA complex that resembles a micro-pre-tRNA. We examined four hook RNAs containing systematically weakened T stems for directing target RNA cleavage by tRNase ZL. As expected, the cleavage efficiency decreased with the decrease in T stem stability, and to our surprise, even the hook RNA that forms no T stem-loop-directed slight cleavage of the target RNA, suggesting that the T stem-loop structure is important but dispensable for substrate recognition by tRNase ZL. To analyze the effect of the T loop on substrate recognition, we compared the cleavage reaction for a micro-pre-tRNA with that for a 12-base-pair double-stranded RNA, which is the same as the micro-pre-tRNA except for the lack of the T loop structure. The observed rate constant value for the double-stranded RNA was comparable with that for the micro-pre-tRNA, whereas the K(d) value for the complex with the double-stranded RNA was much higher than that for the complex with the micro-pre-tRNA. These results suggest that the T loop structure is not indispensable for the recognition, although the interaction between the T loop and the enzyme exists. Cleavage assays for such double-stranded RNA substrates of various lengths suggested that tRNase ZL can recognize and cleave double-stranded RNA substrates that are longer than 5 base pairs and shorter than 20 base pairs. We also showed that double-stranded RNA is not a substrate for the short form of tRNase Z.