Richard C. Ogden

Splicing of yeast tRNA precursors: a two-stage reaction

Soluble extracts of S. cerevisiae splice tRNA precursors which contain intervening sequences. The reaction goes to completion and requires ATP for the production of mature sequence tRNA. In the absence of ATP, half-tRNA molecules accumulate. Similar half-tRNA molecules appear as kinetic intermediates and accumulate if splicing is inhibited with pure, mature tRNA. Half-tRNA molecules have been purified. These half-tRNAs are efficiently ligated in an ATP-dependent reaction that is inhibited by added mature tRNA. The product of ligation is the expected mature sequence tRNA. The excised intervening sequence has also been identified. These results suggest an enzymatic mechanism for splicing which involves two independent steps.

In vitro transcription and processing of a yeast tRNA gene containing an intervening sequence.

A gene for Saccharomyces cerevisiae tRNATrp has been sequenced which contains an intervening sequence of 34 bp (H. S. Kang and J. Abelson, unpublished results). The mutant yeast strain ts-136 accumulates a precursor to tRNATrp which contains mature ends and is colinear with the tRNATrp gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNATrp gene contained on plasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence. The Xenopus extract accurately splices the precursor transcript to mature-sized tRNATrp.

The mechanism of tRNA splicing

Abstract Transfer RNA precursors that contain intervening sequences have been isolated from a yeast mutant. These precursors are substrates in vitro for activities in yeast which excise the intervening sequence and ligate the intermediate tRNA halves. The characteristics of this splicing reaction are discussed.

Enzymatic Removal of Intervening Sequences in the Synthesis of Yeast tRNAs

The phenomenon of noncolinearity between a gene and its mature product has been shown to be a general one in the eukaryotic world. This discovery raised the question of how the cell removes the intervening sequences in the biosynthesis of RNA. Some answers to this question are presented here. The discovery by Hopper et al. (1978) that yeast tRNA precursors accumulate in a mutant strain ( ts 136) has considerably facilitated the study of the RNA splicing reaction. This mutant, isolated by Hutchison et al. (1969), defines the rna1 gene of yeast. It is presumed to be defective in a step in RNA transport from nucleus to cytoplasm. At the nonpermissive temperature, the 35S rRNA precursor accumulates (Hopper et al. 1978), the appearance of mRNA in the cytoplasm is halted, poly(A)-containing RNA accumulates in the nucleus (Shiokawa and Pogo 1974), and a particular subset of tRNA precursors accumulates (Knapp et al. 1978). The separation of those tRNA precursors that accumulate in ts 136 has been accomplished by two-dimensional polyacrylamide gel electrophoresis. A typical two-dimensional separation is shown in Figure 1. Originally the precursor-specific spots were identified by hybridization of the RNA to a set of Escherichia coli recombinant plasmid clones, each of which carries one or more yeast tRNA genes (Beckmann et al. 1977). Five of the RNAs (spots indicated in Fig. 1) hybridized to clones that have been identified as containing genes for tRNA Tyr , tRNA Phe , tRNA 3 Leu , tRNA UCG Ser , and tRNA Trp . These identifications have been subsequently confirmed by RNA sequence analysis. Four other...