I have fused CTD to the GAL4
DNA binding domain and screened mouse cDNA fusion library by two-hybrid method.
Yeast CTD–GAL4 DNA binding domain fusion is capable to activate transcription
in yeast and therefore, it is impossible to use this fusion protein for the
two-hybrid screen. I found that the last 16 repeats of mouse CTD fused
to the GAL4 DNA binding domain were incapable of transcription activation.
I have also found that the same portion of mouse CTD could substitute yeast
wild type CTD and support yeast cell viability. That is why, the two-hybrid
screen with it was functionally relevant.
I have also devised a criteria
for a success of the two-hybrid screen with CTD. The decision was made
to look for proteins that share common CTD-interacting domain. The
rational for this decision was that the conservation of such domain in evolution
was dictated by conservation of the CTD structure as well as its simplicity.
I have performed several two-hybrid screens with mouse embryonic cDNA fusion library. After
analyzing about 2,000,000 transformants I have collected 8 different positive
cDNA clones. Upon their sequence three of them had the CTD-interacting
domain with 90% homology between them and another two
proteins had also 90% conserved CTD-interacting domain of different type.
To investigate the functional relationship between two families of the CTD-interacting
proteins I have cloned the full-length cDNAs of the proteins from each family.
The bioinformatic analysis of the full-length amino acid sequences showed
that the proteins of both families had sequence features similar to an emerging
family of splicing factors. It also suggested the function for the
CTD-interacting proteins was to link transcription and splicing, and to direct
newly synthesized hnRNA to the spliceosomes for further processing.
One of the CTD-interacting proteins itself had about 20 almost identical
repeats with consensus sequence PQPGM (GB#U49058).
About six month after my graduation
yeast protein NrdI was found in the genetic
screen for the suppressors of the foreign insertion in the yeast intron.
Its amino acid sequence showed resemblance to mouse CTD-interacting proteins
and its N-terminal domain interacted with mouse CTD in the two-hybrid assay.