Genetic analysis of the processing of a spliced tRNA

We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutants examined. These enzymes may recognize only limited regions of the precursor RNA. RNA polymerase III behaves as if able to count the number of Us added before termination; and aberrant termination products in two mutants suggest that the secondary structure of the nascent transcript can be very imortant in eukaryotic transcription termination.