Expression of the C-terminal domain of novel human SR-A1 protein: interaction with the CTD domain of RNA polymerase II.
We have recently cloned a new member of the human Ser/Arg-rich superfamily (SR) of pre-mRNA splicing factors, SR-A1. Members of the SR family of proteins have been shown to interact with the C-terminal domain (CTD) of the large subunit of RNA polymerase II, and participate in pre-mRNA splicing. The largest subunit of RNA polymerase II contains at the carboxy-terminus a peculiar repetitive sequence that consists of 52 tandem repeats of the consensus motif Tyr-Ser-Pro-Thr-Ser-Pro-Ser, referred to as the CTD. There is evidence that SR protein splicing factors are involved in cancer pathobiology through their involvement in alternative processing events. The CTD of human SR-A1 protein (aa 1187-1312), containing a conserved CTD-interaction domain and bearing a decahistidine (His10) tag was produced by DNA recombinant overexpression techniques in Escherichia coli from the vector pET16b and it was localized in the periplasmic space. The protein was further purified using a HiTrap chelating column and its circular dichroism spectra indicate that it assumes a defined structure in solution. Performing a pull-down assay we proved that the novel SR-A1 [1187-1312 His10] protein interacts with the CTD domain of RNA polymerase II.
Institute of Physical Chemistry, NCSR Demokritos, 153 10 Ag. Paraskevi Attikis, Greece.
Maria E
ME
Katsarou
Athanasios
A
Papakyriakou
Nikos
N
Katsaros
Andreas
A
Scorilas
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003198:Computer Simulation; D006801:Humans; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; C503566:SCAF1 protein, human; D000068103:Serine-Arginine Splicing Factors; D012319:RNA Polymerase II