S-Space College of Natural Sciences (자연과학대학) Biophysics and Chemical Biology (생물물리 및 화학생물학과) Theses (Master's Degree_생물물리 및 화학생물학과)
The Molecular Mechanism of Rat1-Mediated RNA Polymerase II Transcription Termination
RNA Polymerase II 전사 종결에 관여하는 Rat1의 분자생물학적인 기작 규명에 관한 연구
- 자연과학대학 생물물리 및 화학생물학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 생물물리 및 화학생물학과, 2015. 2. 이준호.
- Transcription termination factors, recruited to RNA Polymerase II (Pol II) C-terminal Domain (CTD), determine the termination process according to the phosphorylation pattern of CTD. Various kinases take parts in the CTD phosphorylation depending on gene length. There are two transcription termination pathways briefly. One is Nrd1-dependent pathway, which is carried out in short genes especially, sn/sno RNA or cryptic unstable transcripts (CUTs). In long genes like cording genes, Rat1, the 5to 3exoribonuclease, carries out Pol II termination with its cofactor, Rai1, and CTD binding protein, Rtt103.
To test the Rat1-dependent termination pathway, I hypothesized specific conditions required for Pol II termination. First, paused Pol II will be more easily terminated by exoribonuclease. Secondly, there will be a limit in the length of nascent RNA to terminate Pol II by Rat1. Lastly, Rat1 might contain other activities except for exonuclease activity. To verify this hypothesis, I designed Pol II pausing condition with various RNA templates, and made rat1 point mutants by PCR mutagenesis. Also, I purified recombinant proteins from E. coli through 4-steps chromatographies and 12 subunits-RNA Polymerase II from S. cerevisiae to use them in vitro system. I performed experiments to figure out molecular mechanism of Pol II termination.
NTP misincorporation enhances Pol II termination by Rat1/Rai1 complex. This is because paused Pol II induced by NTP misincorporation might be vulnerable to processive Rat1/Rai1 complex. And with the longer RNA length, the better Pol II termination by Rat1/Rai1 is achieved. The processivity of Rat1/Rai1 might accumulate along the long RNA.
In addition, I find out various rat1 point mutants showing termination defects in vivo and in vitro test. One of them, rat1 S694A mutant and rat1 E203A/D233A/D235A mutant are lethal in S. cerevisiae. It is true that there are other active domain to be studied for termination in addition to dominant exonuclease activity domain in Rat1. This is because that some mutants, affecting Rai1-interacting domain, show cell viability defects in vivo and termination defects in vitro.
These results imply that Rat1/Rai1 complex is sufficient to terminate Pol II under proper conditions like paused Pol II, long nascent RNA, and undefined specific activities of Rat1. I suggest that these factors might be important for cell viability and Pol II termination.