Small Angle X-ray Scattering study of conformational changes in steroid responsive activator (SRA) RNA upon binding with suppressor protein SHARP
Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Thomas C. Leeper
Dr. Kimberly Cortes
Dr. Huggins Msimanga
Differentiation and proliferation are known to be influenced by steroid stimulation. The steroid receptor activator (SRA) RNA is a large RNA transcript indicated to be an epigenetic regulatory component in the steroid receptor coactivator-1 (SRC-1) acetyltransferase complex. It is needed for a response to steroid hormones by tumor cells. However, this activity is hindered by suppressive RNA binding proteins like the SMRT/HDAC1-associated repressor protein (SHARP), which binds the STR7 domain of SRA RNA through its four RNA recognition motifs (RRMs); it is not fully understood how SHARP executes this role as there are no SRA RNA-SHARP structures available for study. Traditional methods have determined the secondary structure of STR7 and the Leeper group used NMR to determine the structures of SHARP RRMs, but these techniques have shown to be limited in the case of SRA RNA-SHARP complexes because of its size and flexibility.
In this study, SHARP2 was purified and complexed with STR7 RNA synthesized from oligonucleotides and T7 RNA polymerase. Small angle X-ray scattering was then used to investigate and compare the structures of the three-dimensional conformations of SRA RNA-SHARP2 complexes to the free-SHARP2 and the free-STR7 RNA structures to better understand how SHARP carries out its function and thereby improve therapeutic treatments for cancer. The data obtained showed structural changes in the RNA and an increase in the radius of gyration, which supports the hypothesis that SHARP RRM 2 contributes to a reduction in the SRA RNA coactivator activity by remodeling the RNA structure while the remaining SHARP RRMs may act simply as recruitment modules. Therefore, if we can determine how SHARP suppresses SRA RNA activity, then small molecules can be designed to counter SRA RNA activity and improve cancer therapeutics.