A Life with Yeast Molecular Biology - Prof. Dr. Horst Feldmann

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H. FELDMANN
Besides this structural work I became interested in the
characterization of precursors to tRNA in yeast as a model system.
tRNA precursors had already been studied in E. coli and phage T4
[23,24] and Sidney Altman and collaborators had detected that
endonuclease P was necessary to produce mature tRNA from its
precursors [25], but no details were known in eukaryotes [26]. We
used gel electrophoresis of ‘‘soluble’’ RNA preparations of yeast
cells pulse-labeled with high doses of [ 32 P] phosphate and found
that some distinct bands migrating slower than mature tRNA
appeared. The smaller of these products contained minor amounts
of the tetranucleotide TCCGp characteristic for tRNA but only
traces of other minor nucleotides, while the larger of these
products contained the tetranucleotide UUCGp but no minor
nucleotides. Thus we concluded that these bands represented
precursors to tRNA. This was confirmed by incubating material
isolated from several of these gel bands in vitro in a yeast cell
lysate, which yielded mature tRNA in kind of a two-step process
[27]. Soon after, Goodman, Olson, and Hall reported on the first
yeast tRNA gene to have an intervening sequence [28].
In conjunction with our efforts to identify the precursors to
tRNA, we developed a two-dimensional gel electrophoretic system
that enabled us to reproducibly map 40–50 individual tRNA
species including isoacceptors from yeast, as well as individual
precursors [29]. tRNAs were identified by (i) co-electrophoresis
of purified [ 32 P] tRNAs with non-labeled bulk tRNA;
(ii) comparison of patterns derived from pure tRNAs with bulk
tRNA; (iii) fingerprinting of spots from pure [ 32 P] tRNA species;
(iv) electrophoresis of bulk tRNA charged with one [ 3 H]- or [ 14 C]amino
acid, whereby the aminoacyl tRNA was stabilized prior to
electrophoresis by transforming the amino group into a 100-fold
more stable OH-group. We realized that this technique of high
resolution capacity – among other applications – was not only
helpful to islolate specific yeast tRNAs but to identify the amino
acid accepted by them, likewise also for determining the specific
tRNAs contained in a tRNA population from any organism. One
example is kindly mentioned by Guy Dirheimer, whom I met for
the first time in the 60’s, in volume 44 of these Personal
Recollections [30]. He sent Jean Weissenbach to my lab to learn
the details of the procedure. They applied it to separate and
isolate the mitochondrial tRNAs from yeast and started sequencing
of several of those (e.g. [31]), a subject the Strasbourg lab