Endonucleolytic cleavage of a long 3'-trailer sequence in a nuclear yeast suppressor tRNA

Transcripts of Saccharomyces cerevisiae nuclear tRNA genes are normally terminated within a few nucleotides of the tRNA coding region, in contrast to mitochondrially encoded tRNAs, which are contained within polycistronic transcripts and thus require 3'-processing by mitochondrial endonucleases. We show that 3'-processing activities capable of removing artificially extended 3'-trailer sequences from some tRNA substrates are also present in the yeast nucleus. Correct 3'-processing in vivo resulted in the formation of functional suppressor tRNA. The 3'-processing activities were also identified in vitro through analysis of transcription-processing products in cell-free yeast S-100 extracts. Comparison of several pre-tRNA substrates showed that the tRNA structure played a major role in determining the processability of a substrate but that the nature of the 3'-trailer sequence also modulated the rate of 3'-processing. Pre-tRNA containing mitochondrial tRNA(Val) sequence was a good substrate for in vitro processing, independent of its 3'-trailer. A 200-nt-long pre-tRNA, encoding the nuclear SUP4 tRNA gene and a mitochondrial 3'-trailer, was processed in yeast S-100 extract in a multistep pathway into mature-sized tRNA(Tyr). Part of the 3'-processing was due to an endonuclease which cleaved near or precisely at the 3'-end of the coding region of the tRNA. A short sequence around this endonucleolytic 3'-cleavage site was crucial for the formation of active suppressor tRNA in vivo. A 9-nt-long sequence motif derived from the mitochondrial 3'-trailer allowed processing, while sequences derived from lacZ or pBR322 DNA were processed neither in vitro nor in vivo.