This project was to address a function of the C-terminal domain
of eukaryotic RNA polymerase II (CTD) using methods of yeast genetics.
Corden’s lab was competing with Rick Young’s lab at MIT. R.Young's
group has shown that the deletion of 18 out of 25 CTD repeats in yeast polymerase
II caused the cold sensitive phenotype and that the deletion of 19 CTD repeats
is lethal. Marilyn West in Corden’s lab has
constructed CTD substitution mutants in which putative CTD phosphorylation
sites were substituted for alanines to mimic dephosphorylated state or glutamic
acids to mimic phosphorylated state (YSPTSPS to YA/EPTSPS and YSPTSPS to YSPTA/EPS mutants). She also found that such
substitutions were lethal and yeast cells could not live with such mutated
CTDs.
My goal was to find the mutant yeast cells that actually can grow with such
substituted CTDs by acquiring a suppressing mutation(s). The suppressor
mutants, that I found, were extremely slow growing (colony were visible
only after 5-7 days of growth). A technique that I used for isolating
and cloning a suppressor by the genetic complementation involved plasmid shuffling
method developed in Jeff Boeke’s lab.
In brief, yeast strain was constructed that had a deletion of the wild type
large subunit RNA polymerase II gene (RPB1). Wild type
RPB1 gene was maintained in this strain on URA3 plasmid.
The rpb1 genes carrying different CTD mutants were introduced into
this strain on LEU2 plasmid. URA3 plasmid can be counter selected
on the medium containing 5-FOA. This allowed the testing for viability of
different CTD mutants as well as the isolating of their suppressors by selecting
for 5-FOAr colonies. To enhance a selection power of the
system a second counter selectable marker, CYH2, was introduced into
URA3 plasmid carrying wild type RPB1 gene. Thus, the
double counter selection with both 5-FOA and cycloheximide was used to isolate
suppressors of the CTD mutant. One of the 5-FOAr,cyhr
strains was used to clone a suppressor gene from yeast genomic library.
The library plasmids that restored strain sensitivity to 5-FOA and
cycloheximide were recovered and sequenced.
As a result of this effort, I have isolated a novel yeast gene with unknown
function. In parallel with my work R.Young’s graduate student at MIT
cloned the suppressor of cold sensitive CTD deletion. As it turned
out he cloned the same gene. R. Young named this new gene SRB9
and I called it SCA1 (GB# P38931). I have
also found that SCA1 gene was non-essential for yeast viability and
that SCA1 deletion is enough to suppress lethal CTD substitutions.
This fact suggested that SCA1 suppression was a bypass effect.