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菠菜叶绿体转移RNA基因启动子的鉴定及突变分析

Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene.

作者信息

Gruissem W, Zurawski G

出版信息

EMBO J. 1985 Jul;4(7):1637-44. doi: 10.1002/j.1460-2075.1985.tb03831.x.

DOI:10.1002/j.1460-2075.1985.tb03831.x
PMID:2992936
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC554398/
Abstract

A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements. Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNAs. Transcription of the spinach chloroplast tRNA2Met gene (trnM2) in vitro requires 5' upstream DNA sequences. Deletion of 5' DNA sequences with exonuclease Bal31 was used to establish the 5' boundary of the promoter region. This boundary is part of a DNA sequence with partial homology to the prokaryotic -35 region. Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic -10 region. We used synthetic oligonucleotides fused to trnM2 5' deletion mutants to create insertions, deletions and base substitutions in these regions. Internal deletion mutants demonstrated that the -10 promoter element is also required for transcription in vitro. The arrangement of DNA sequences recognised by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.

摘要

从纯化的菠菜叶绿体中提取转录物，用于测试叶绿体DNA序列作为启动子元件的功能。叶绿体tRNA基因在提取物中由可溶性RNA聚合酶正确转录，前体分子被加工成成熟的tRNA。菠菜叶绿体tRNA2Met基因（trnM2）的体外转录需要5'上游DNA序列。用核酸外切酶Bal31缺失5' DNA序列来确定启动子区域的5'边界。该边界是与原核生物-35区域具有部分同源性的DNA序列的一部分。在该序列下游17个碱基对处出现一个与原核生物-10区域同源的DNA序列。我们使用与trnM2 5'缺失突变体融合的合成寡核苷酸在这些区域产生插入、缺失和碱基替换。内部缺失突变体表明，-10启动子元件对于体外转录也是必需的。叶绿体RNA聚合酶识别的DNA序列排列类似于原核生物启动子组织。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/9fee5482ff07/emboj00272-0029-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/7275f7cf5abc/emboj00272-0026-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/6d7f854cfd61/emboj00272-0028-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/9fee5482ff07/emboj00272-0029-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/7275f7cf5abc/emboj00272-0026-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/6d7f854cfd61/emboj00272-0028-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/22f0/554398/9fee5482ff07/emboj00272-0029-a.jpg

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本文引用的文献

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Proc Natl Acad Sci U S A. 1982 Dec;79(24):7699-703. doi: 10.1073/pnas.79.24.7699.
2
Structures of the genes for the beta and epsilon subunits of spinach chloroplast ATPase indicate a dicistronic mRNA and an overlapping translation stop/start signal.菠菜叶绿体 ATP 酶β和ε亚基基因的结构表明，该基因是一个双顺反子 mRNA，且存在一个翻译终止/起始信号重叠。
Proc Natl Acad Sci U S A. 1982 Oct;79(20):6260-4. doi: 10.1073/pnas.79.20.6260.
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豌豆叶绿体 tRNA（Lys）（UUU）基因：内含子基因的转录和分析。
Photosynth Res. 1988 Jul;17(1-2):7-22. doi: 10.1007/BF00047679.
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Using bacteria to analyze sequences involved in chloroplast gene expression.利用细菌分析参与叶绿体基因表达的序列。
Photosynth Res. 1988 Jan;19(1-2):7-22. doi: 10.1007/BF00114566.
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9
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