Transcription and Processing of Transfer RNA in S. cerevisiae

Citation

Reyes, Vicente Mendoza (1988) Transcription and Processing of Transfer RNA in S. cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3md6-cn17. https://resolver.caltech.edu/CaltechTHESIS:03112013-111743236

Abstract

A collection of studies on tRNA transcription and splicing in the yeast S. cerevisiae are presented. These studies employ a combination of recombinant DNA, oligonucleotide-directed mutagenesis and in vitro synthetic technologies applied using the reverse genetics approach. Chapter I introduces the reader to these topics. In Chapter II, an attempt to solve the puzzle of tandem tRNA gene transcription in yeast is described. This tandem, the S. cerevisae tRNA^Arg-tRNA^Asp gene pair, is transcribed solely by use of the upstream gene promoter signals, giving rise to a dimeric precursor which is processed into two mature tRNA molecules, much like prokaryotic systems. A collection of specific point and deletion mutations were constructed to answer the question of why the downstream tRNA^Asp gene is apparently inactive. Our results show that it is so only in this configuration; the tRNA^Arg and spacer sequences, which constitute its upstream flanking sequences in this arrangement, seem inhibitory to the independent activity of the tRNA^Asp gene. Taken together, these results emphasize the importance of flanking regions in eukaryotic tRNA gene transcription. In Chapter III, the construction and characterization of a heterologous system for the in vitro synthesis of pre-tRNA are presented. A strong bacteriophage T7 promoter was fused to a S. cerevisiae pre-tRNA^Phe gene. We show that pre-tRNA^Phe is synthesized efficiently from this system by the cognate T7 RNA polymerase, and that this RNA has the correct sequence, mature terminii, and is spliced efficiently and accurately by our in vitro splicing system, which consists of highly purified tRNA splicing endonuclease and ligase enzymes. In Chapter IV, an extensive investigation on tRNA splicing substrate specificity is described. A collection of 15 carefully-designed mutant pretRNA^Phe genes were constructed, and then transcribed and analyzed as above. We find that the endonuclease recognizes two highly conserved, surface residues in pre-tRNA^Phe-U8 and C56-and probably contacts these bases during the splicing reaction. We also find that splice site selection by this enzyme is a function of the length of the anticodon stem, and thus proceeds by a simple distance measurement mechanism. We have evidence that this measuring process commences in the thoracic region of the pre-tRNA, where the endonuclease probably binds. Finally, we demonstrate that the highly conserved purine residue 3' proximal to the anticodon may be important for cleavage at the nearby 5' splice site.

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