東華大學圖書館 |

The group I ribozyme from the chloroplast rRNA gene of Chlamydomonas reinhardtii: Kinetic and structural analysis of the divalent metal requirement and specific interactions with manganese (II).

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	The group I ribozyme from the chloroplast rRNA gene of Chlamydomonas reinhardtii: Kinetic and structural analysis of the divalent metal requirement and specific interactions with manganese (II)./
作者:	Kuo, Tai-Chih.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 1998,
面頁冊數:	226 p.
附註:	Source: Dissertations Abstracts International, Volume: 60-06, Section: B.
Contained By:	Dissertations Abstracts International60-06B.
標題:	Botany. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9905773
ISBN:	9780599033009

The group I ribozyme from the chloroplast rRNA gene of Chlamydomonas reinhardtii: Kinetic and structural analysis of the divalent metal requirement and specific interactions with manganese (II).
Kuo, Tai-Chih.

The group I ribozyme from the chloroplast rRNA gene of Chlamydomonas reinhardtii: Kinetic and structural analysis of the divalent metal requirement and specific interactions with manganese (II). - Ann Arbor : ProQuest Dissertations & Theses, 1998 - 226 p.

Source: Dissertations Abstracts International, Volume: 60-06, Section: B.

Thesis (Ph.D.)--The University of Texas at Austin, 1998.

This item must not be sold to any third party vendors.

Cr.LSU is a self-splicing group I intron in the chloroplast 23S rRNA gene of the green alga Chlamydomonas reinhardtii. In vitro, Cr.LSU catalyzes its own excision from precursor RNA (pre-RNA), which is coupled to exon ligation. The self-splicing of Cr.LSU can be achieved with Mg$\\sp{2+}$ and a guanosine nucleotide (G). The ribozyme is highly specific for guanosine. The requirement for a divalent metal in the structure and function of Cr.LSU, however, has not been investigated extensively, and was one of the goals of the present research. Also, quantitative kinetic data were lacking for Cr.LSU activity. In order to better characterize the structure and function of Cr.LSU, a smaller pre-RNA that self-spliced efficiently was needed. I found that certain sequences in the 5'-exon reduced splicing efficiency. By shortening the 5'-exon and 3'-exon sequences, and removing a nonessential intron sequence, a 448-nt RNA, 23S.5, was generated. 23S.5 pre-RNA self-spliced efficiently in the absence of monovalent salts. Quantitative kinetic analysis indicated that the pre-RNA population was heterogeneous; $\\sim$80% reacted rapidly and $\\sim$20% reacted slowly. The kinetic parameters, K$\\rm \\sb M\\sp G$ (G concentration for half maximal rate of self-splicing) and $k\\sb{cat}$ (maximal rate of intron splicing), for the highly reactive fraction were determined and found to be $\\sim$28 $\\mu$M and 1.0 min$\\sp{-1}$, respectively. The self-splicing of 23S.5 pre-RNA was supported by Mg$\\sp{2+}$, but was inhibited by Mn$\\sp{2+}$. The binding of at least two Mg$\\sp{2+}$ were required for full activity, whereas full inhibition required the uptake of at least three Mn$\\sp{2+}$. The structure and folding of the Cr.LSU intron were probed with Fe$\\sp{2+}$-EDTA and a modified version of 23S.5 pre-RNA, 23S.5$\\Delta$G$\\sb{\\rm b}$, which lacks the last 3 nt of the intron and the 3'-exon. This RNA was necessary to prevent cyclization of the excised intron. The results indicated that the core of the Cr.LSU intron is not as well protected as the Tetrahymena and phage group I ribozymes. Comparisons of the activity and Fe$\\sp{2+}$-EDTA cleavage/protection patterns as a function of Mg$\\sp{2+}$ concentration suggested that folding of the core was limiting, at least in terms of the Mg$\\sp{2+}$ requirement. At pH values higher than 6.5, Mn$\\sp{2+}$ caused site-specific cleavages of the Cr.LSU intron. Specific cleavage of the 659-nt 23S.3 RNA occurred at six sites (I-VI), all of which contained the sequence GAAA. Cleavage was between the G and AAA, and only GAAA sequences were specifically cleaved. Cleavage occurred much more rapidly at two "major" sites compared to four "minor" sites. Plots of cleavage rate vs. Mn$\\sp{2+}$ concentration for cleavage at the major sites gave hyperbolic saturation curves, indicative of specific Mn$\\sp{2+}$ binding. High concentrations of Mg$\\sp{2+}$ inhibited Mn$\\sp{2+}$-dependent cleavage at the sites, suggesting that these sites may also bind Mg$\\sp{2+}$. Consistent with this view, Mg$\\sp{2+}$ alone induced cleavage at site III. These data indicate that GAAA can specify metal binding, and that binding can include Mg$\\sp{2+}$ as well as Mn$\\sp{2+}$. (Abstract shortened by UMI.).

ISBN: 9780599033009Subjects--Topical Terms:

516217
Botany.
Subjects--Index Terms:

Chlamydomonas reinhardtii

The group I ribozyme from the chloroplast rRNA gene of Chlamydomonas reinhardtii: Kinetic and structural analysis of the divalent metal requirement and specific interactions with manganese (II).
LDR:04705nmm a2200409 4500 001 2344749
005 20220531065747.5
008 241004s1998 ||||||||||||||||| ||eng d
020 $a 9780599033009
035 $a (MiAaPQ)AAI9905773
035 $a AAI9905773
040 $a MiAaPQ $c MiAaPQ
100 1 $a Kuo, Tai-Chih. $3 3683554
245 1 4 $a The group I ribozyme from the chloroplast rRNA gene of Chlamydomonas reinhardtii: Kinetic and structural analysis of the divalent metal requirement and specific interactions with manganese (II).
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 1998
300 $a 226 p.
500 $a Source: Dissertations Abstracts International, Volume: 60-06, Section: B.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Herrin, David L.
502 $a Thesis (Ph.D.)--The University of Texas at Austin, 1998.
506 $a This item must not be sold to any third party vendors.
506 $a This item must not be added to any third party search indexes.
520 $a Cr.LSU is a self-splicing group I intron in the chloroplast 23S rRNA gene of the green alga Chlamydomonas reinhardtii. In vitro, Cr.LSU catalyzes its own excision from precursor RNA (pre-RNA), which is coupled to exon ligation. The self-splicing of Cr.LSU can be achieved with Mg$\\sp{2+}$ and a guanosine nucleotide (G). The ribozyme is highly specific for guanosine. The requirement for a divalent metal in the structure and function of Cr.LSU, however, has not been investigated extensively, and was one of the goals of the present research. Also, quantitative kinetic data were lacking for Cr.LSU activity. In order to better characterize the structure and function of Cr.LSU, a smaller pre-RNA that self-spliced efficiently was needed. I found that certain sequences in the 5'-exon reduced splicing efficiency. By shortening the 5'-exon and 3'-exon sequences, and removing a nonessential intron sequence, a 448-nt RNA, 23S.5, was generated. 23S.5 pre-RNA self-spliced efficiently in the absence of monovalent salts. Quantitative kinetic analysis indicated that the pre-RNA population was heterogeneous; $\\sim$80% reacted rapidly and $\\sim$20% reacted slowly. The kinetic parameters, K$\\rm \\sb M\\sp G$ (G concentration for half maximal rate of self-splicing) and $k\\sb{cat}$ (maximal rate of intron splicing), for the highly reactive fraction were determined and found to be $\\sim$28 $\\mu$M and 1.0 min$\\sp{-1}$, respectively. The self-splicing of 23S.5 pre-RNA was supported by Mg$\\sp{2+}$, but was inhibited by Mn$\\sp{2+}$. The binding of at least two Mg$\\sp{2+}$ were required for full activity, whereas full inhibition required the uptake of at least three Mn$\\sp{2+}$. The structure and folding of the Cr.LSU intron were probed with Fe$\\sp{2+}$-EDTA and a modified version of 23S.5 pre-RNA, 23S.5$\\Delta$G$\\sb{\\rm b}$, which lacks the last 3 nt of the intron and the 3'-exon. This RNA was necessary to prevent cyclization of the excised intron. The results indicated that the core of the Cr.LSU intron is not as well protected as the Tetrahymena and phage group I ribozymes. Comparisons of the activity and Fe$\\sp{2+}$-EDTA cleavage/protection patterns as a function of Mg$\\sp{2+}$ concentration suggested that folding of the core was limiting, at least in terms of the Mg$\\sp{2+}$ requirement. At pH values higher than 6.5, Mn$\\sp{2+}$ caused site-specific cleavages of the Cr.LSU intron. Specific cleavage of the 659-nt 23S.3 RNA occurred at six sites (I-VI), all of which contained the sequence GAAA. Cleavage was between the G and AAA, and only GAAA sequences were specifically cleaved. Cleavage occurred much more rapidly at two "major" sites compared to four "minor" sites. Plots of cleavage rate vs. Mn$\\sp{2+}$ concentration for cleavage at the major sites gave hyperbolic saturation curves, indicative of specific Mn$\\sp{2+}$ binding. High concentrations of Mg$\\sp{2+}$ inhibited Mn$\\sp{2+}$-dependent cleavage at the sites, suggesting that these sites may also bind Mg$\\sp{2+}$. Consistent with this view, Mg$\\sp{2+}$ alone induced cleavage at site III. These data indicate that GAAA can specify metal binding, and that binding can include Mg$\\sp{2+}$ as well as Mn$\\sp{2+}$. (Abstract shortened by UMI.).
590 $a School code: 0227.
650 4 $a Botany. $3 516217
650 4 $a Molecular biology. $3 517296
653 $a Chlamydomonas reinhardtii
653 $a Chloroplast rRNA
653 $a Divalent metal
653 $a Iron
653 $a Magnesium
653 $a Manganese
653 $a Ribozyme
690 $a 0309
690 $a 0307
710 2 $a The University of Texas at Austin. $3 718984
773 0 $t Dissertations Abstracts International $g 60-06B.
790 $a 0227
791 $a Ph.D.
792 $a 1998
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9905773