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            I believe that  this project is, by this day, my biggest scientific achievement.   Looking from the point of view of today’s developments in the two-hybrid screen method the project was rather simple.  I have fused CTD to the GAL4 DNA binding domain and screened mouse cDNA fusion library.  However,  there were two major obstacles for doing the two-hybrid screen with the CTD.
         First, is the fact that the yeast CTD–GAL4 DNA binding domain fusion is quite capable to activate transcription in yeast and therefore, it is impossible to use this fusion protein for two-hybrid screen.  Based on his knowledge that the last 16 repeats of mouse CTD can not support RNA polymerase II function in vivo, Jeff Corden suggested to fuse it to the GAL4 DNA binding domain.  I was so much curious in doing the two-hybrid screen that in spite of my occupancy with analysis of genetic suppressors I have made this fusion and found that the mouse CTD repeats did not activate transcription1We were cleared to do two-hybrid screen !.
        The second obstacle was to come up with the criteria, which can be used to evaluate found CTD interacting proteins.  It is clear, both intuitively and from reading the more recent literature, that the two-hybrid screen provides user with both functionally relevant and irrelevant interactions 2.
        To develop the criteria for the successful two-hybrid with the CTD I looked once again at the CTD structure.  I came up with the same conclusion as most researchers did: CTD had very simple structure - one heptapeptide repeated multiple times.  I then assumed that the CTD had multiple protein partners in vivo, that physically interact with it.  I thought that the simplicity of the CTD structure must dictate the conservation of a CTD-interacting domain 3.    So, I have decided to keep screening for CTD interactors until I would find proteins that had similar CTD-interacting domains.  In my first screen of mouse embryonic cDNA fusion library I have obtained only about 500,000 transformants that gave me five different positive clones.  None of them had any similarity to another by sequence.  Since 500,000 transformants did not saturate the library screen, I performed another screen and obtained about 2,000,000 transformants.  They yielded  8 different positives.  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.  Though, I believed that I had achieved total success in the two-hybrid screen there was one question remaining: why there were two families of the CTD-interacting proteins and which of them was real ?
        To obtain the answer I decided to start by cloning the full-length cDNAs of the proteins from each family.  The results again exceeded my expectations:  the analysis of their full-length aminoacid sequences showed that proteins of both families had sequence features similar to the emerging family of splicing factors.  Thus, their sequences suggested the function for the CTD-interacting proteins:  to link transcription and splicing, to direct newly synthesized hnRNA to the spliceosomes for further processing.  As an additional reward of this effort one of the CTD-interacting proteins itself had about 20 almost identical repeats with consensus sequence PQPGM (GB#U49058).  Based on the success of the two-hybrid screen with repetative CTD, I hope everyone understands what kind of experiment must be done with this repetitive domain.
        I have spent the rest of my Ph.D. education in the attempts to graduate as soon as possible and to find the CTD interacting proteins in yeast.  All my attempts had failed.  About six month after my graduation yeast CTD interacting protein NrdI was found in the genetic screen for the suppressors of the foreign insertion in the yeast intron.
 

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1.     I think now that the inability of mouse CTD to activate transcription has nothing to do with its inability to support viable RNA polymerase II in mouse cells.  In fact, I later showed that the same portion of mouse CTD could substitute yeast CTD in yeast cells.  Yeast are quiet viable and happy with mutated RNA polymerase II, that has the portion of mouse CTD in stead of its wild type one. (back to the main reading).
        Yeast CTD is capable to activate transcription, probably, because it has an acidic polypeptide structure.   Many acidic helical polypeptides can be transcription activators when fused to GAL4 DNA binding domain.  The last 18 repeats of mouse CTD have multiple positively charged amino acid substitutions in the last position of the consensus YSPTSPS repeat sequence.  Most of them have the consensus sequence: YSPTSPK.  It is likely, that these positively charged substitutions neutralize CTD acidity and block transcription activation.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

2.    This problem is different from the problem of sorting out weak, non-specific interactions during the two-hybrid screen.   I believe that the current developments in the technique allow accurate user to eliminate weak interactions.  Yet, there are a lot of strong protein-protein interactions that are physiologically unimportant and possibly never occur inside the living cell.  Proteins are designed by nature to interact with each other and other biological macromolecules.  Their function is to interact and form the structure underlying the living system (cell or multicellular organism) and to change the living structure in response to changes in the environment.  So how can the experimentalist distinguish functional protein-protein interactions from the list of the interactions provided by the two-hybrid screen ?   The answer is obviously specific for every protein but the two-hybrid screen with CTD provides an example of solving this problem (back to the main reading)
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

3.        If the part of the protein function is to interact with CTD  why “reinvent the wheel” and come up with new CTD-interacting domain each time the new CTD partner appears in evolution ? (back to the main reading)
 
 















Some old and fresh ideas about functions of CTD interacting proteins (CTDiP).

Disclaimer:  the ideas described below are not polytically correct and not accepted by majority of scientific community.  Since I do not have any opportunity to prove or disprove them experimentally these statements must be considered as hypothesis only or as a fruit of my imagination.  By proceeding past this page you agree with affirm acceptance to the following disclaimer:

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1.  Link transcription in vivo to hnRNA processing and mRNA splicing by directing newly synthesized hnRNA to spliceosomes.

2.  The existence of a family of CTDiPs in mammalian genome suggests that individual hnRNAs can be directed to different pathways for further processing.  Hence, the targeting of mRNA transport in cell cytosole can be determined by the type of CTDiP at the transcription complex assembled on a gene.

3. The discovery of CTDiPs provoces a new paradigm: in eukaryots there may be no such thing as transcription activation, but rather an activation of mRNA maturation pathway by recruiting CTDiP to transcription complex.

4.  An in vivo experiment to prove that CTD function in linking transcription and mRNA maturation pathway:  it was shown that RNA polymerase III can not direct hnRNA to splicing and polyadenilation pathaways (      ).  In brief, the normally polII transcribed gene with intron, which transcription is driven by artificial polIII promotor, produces RNA that is not spliced and polyadenilated.