RNA recognition by the protein component of Escherichia coli RNase P = 대장균 RNase P의 단백질 성분에 의한 RNA 인식

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dc.contributor.advisorLee, Young-Hoon-
dc.contributor.advisor이영훈-
dc.contributor.authorPark, Bo-Hyun-
dc.contributor.author박보현-
dc.date.accessioned2011-12-13T04:28:19Z-
dc.date.available2011-12-13T04:28:19Z-
dc.date.issued2000-
dc.identifier.urihttp://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=157929&flag=dissertation-
dc.identifier.urihttp://hdl.handle.net/10203/31518-
dc.description학위논문(박사) - 한국과학기술원 : 화학과, 2000.2, [ ix, 110 p. ]-
dc.description.abstractRibonuclease P (RNase P) is a tRNA biosynthetic enzyme that participates in the 5`` terminal maturation of tRNAs. In Escherichia coli, it is composed of a catalytic RNA subunit and a protein cofactor, called M1 RNA and C5 protein, respectively. The presence of the C5 protein brings about a variety effects on the RNase P catalysis, whereas M1 RNA can cleave the precursor of tRNAs, under nonphysiological high concentration of salts, in the absence of the protein subunit in vitro. For molecular analysis of the C5 protein function, the protein was obtained in a hybrid form fused to the carboxyl terminus of maltose-binding protein (MBP). The fusion protein, MBP-C5, was easily overproduced in E. coli cells and did not show toxicity for the cell viability that was observed in the overproduction of native C5 protein. The fusion C5 protein was readily purified in a single affinity chromatography using amylose resin. The purified MBP-C5 fusion protein was highly soluble at various working buffers. The functional activity of the fusion protein was tested by its ability to stimulate the activity of M1 RNA in low-ionic strength buffer in vitro and to suppress the growth defect of the thermosensitive rnpA49 mutant strain at nonpermissive temperatures. For in vitro catalytic reaction, authentic M1 RNA, 377-nt in length, was constructed by site-directed mutagenesis under SP6 RNA promoter, and the precursor of $tRNA^{Phe}$ was also constructed as the substrate. Using the fusion system of MBP-C5 protein, four deletion mutants of C5 protein were constructed (C5Δ33, C5Δ72, C5Δ80, and C5Δ106). The smallest mutant C5Δ33 carrying only the proposed unique RNA-binding motif (RNR) did not show the ability to restore the enzymatic activity of M1 RNA. However, they were able to stimulate M1 RNA when either the N- or C-terminal region of C5 protein was combined to the RNR motif as in C5Δ72 or C5Δ80. These results suggest that the RNR motif and the adjacent suggested cleft domain in the C5 ...eng
dc.languageeng-
dc.publisher한국과학기술원-
dc.subjectC5 protein-
dc.subjecttRNA-
dc.subjectSELEX-
dc.subjectRNase P-
dc.subjectin vitro selection-
dc.subject시험관내 선별법-
dc.subjectC5 단백질-
dc.subjecttRNA-
dc.subjectSELEX-
dc.subjectRNase P-
dc.titleRNA recognition by the protein component of Escherichia coli RNase P = 대장균 RNase P의 단백질 성분에 의한 RNA 인식-
dc.typeThesis(Ph.D)-
dc.identifier.CNRN157929/325007-
dc.description.department한국과학기술원 : 화학과, -
dc.identifier.uid000935141-
dc.contributor.localauthorLee, Young-Hoon-
dc.contributor.localauthor이영훈-
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CH-Theses_Ph.D.(박사논문)
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