Conformational transition required for efficient splicing of transcripts from hybrid lambda promoter yeast tRNA gene fusion.

Abstract

Fusion of a prokaryotic promoter to a yeast tRNA gene provides a means for uncoupling analyses of mutations affecting splicing from requirements for transcription and other processing steps. For this purpose, a phage lambda promoter was fused to the Saccharomyces cerevisiae tRNATyr(SUP3a) coding sequence. This fusion allows the synthesis of an end-mature precursor by in vitro transcription with Escherichia coli RNA polymerase. This precursor was accurately spliced by purified yeast endonuclease and ligase fractions. However, both the initial rate and the extent of the endonuclease cleavage reaction were reduced in comparison to those for substrates produced by yeast RNA polymerase III. Efficient splicing could be restored in a magnesium- and temperature-dependent renaturation step, suggesting a conformational transition was required. Enzymatic solution structure probing of transcripts from wild-type and intron-variant templates revealed that the essential conformational transition involved a segment of the tRNA-like portion of the precursor. These results (1) suggest that the primary sequence of the precursor alone may not be sufficient to ensure formation of the active conformer during synthesis, (2) provide direct evidence that endonuclease recognizes mature tRNA-like structure in the precursor, and (3) suggest a general caution for the use of semisynthetic transcripts in RNA processing reactions. Potentially, transcription and processing of tRNATyr in yeast may provide a useful paradigm for examining active control of conformation in RNA biosynthesis.