Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.
Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.
复制标题
烟草核编码叶绿体核糖体蛋白 L27：cDNA 序列以及 mRNA 和基因分析。
DOI：
10.1021/bi00144a028
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Bourque,DP
中科院分区：
文献类型：
--
作者：
Elhag,GA;Bourque,DP
1992年4月27日摘要：烟草（Nicotiana tabacum cv.在表达载体Xgtl 1中构建了小哈瓦那叶片cDNA文库.免疫学和核酸杂交筛选产生了几个cDNA编码的M19 641前体的M，14 420成熟蛋白，这是同源的大肠杆菌核糖体蛋白L27。一个cDNA（L27-1; 882个核苷酸长）含有104 bp的5 ′-非编码序列、51个转运肽密码子、128个预测的成熟L27多肽密码子和241 bp的3 ′-非编码序列，包括聚（A）29尾。将A/β-半乳糖苷酶-L27融合蛋白结合到硝酸纤维素滤膜上，表达，并用作亲和基质以从用50 S叶绿体核糖体蛋白免疫的兔抗血清中纯化抗L27蛋白的单特异性抗体。使用这种单特异性抗体，蛋白质L27中确定的HPLC纯化的烟草叶绿体核糖体50 S亚基蛋白。通过HPLC纯化的L27蛋白的部分测序证实了成熟L27蛋白的预测氨基末端。成熟L27蛋白与枯草芽孢杆菌、大肠杆菌、枯草芽孢杆菌和大肠杆菌的L27型核糖体蛋白的氨基酸同源性分别为66%、61%、56%和48%。coli、嗜热脂肪芽孢杆菌（Bacillus stearo-thermophilus）和酵母线粒体（MRP 7）的同源重叠区。烟草叶绿体核糖体蛋白L27的转运肽与MRP 7线粒体靶向序列有41%的氨基酸序列相似性。烟草叶绿体L27蛋白还具有40个氨基酸长的羧基末端延伸（与其细菌对应物相比），其类似于酵母MRP 7的相应部分。烟草L27的羧基端有一个不寻常的富含半胱氨酸的序列（CFCCC），其功能未知。北方印迹分析显示一条大小（0.85-0.90 kb）对应于全长L27 cDNA的mRNA条带。两个烟草核糖体蛋白L27的cDNA编码序列完全相同，但3 ′端非编码序列不同。L27 cDNA探针与烟草基因组DNA的限制性内切酶酶切片段和克隆的基因组片段杂交，进一步证明了烟草L27存在不止一个基因。核糖体是存在于所有细胞中的蛋白质合成细胞器。细菌核糖体的结构和功能已被深入研究（Lindahl& Zengel，1986; Gourse等人，1986年）。最近的研究已经开始揭示关于真核核糖体的结构和功能的细节（Planta等人，1986; Warner等人，1986; Wool等人，1990; Amaldi等人，1989; Subramanian等人，1990年）。与细菌相反，植物细胞在细胞质、叶绿体和叶绿体中维持多种明显不同类型的核糖体（Boynton et al. 1980年）。这三种蛋白质合成系统在它们的核糖体亚基的质量和密度、它们的组分蛋白质的数量以及大小核糖体亚基和小核糖体亚基的rRNA的大小和序列同源性程度方面不同（Boynton等人，1980;卡佩尔和布尔克，1982;邦汉姆-史密斯和布尔克，1990）。编码叶绿体核糖体蛋白的基因分布在叶绿体和核基因组之间。21个核糖体蛋白基因存在于烟草的叶绿体基因组中（Shinozaki et al.，1986; Yokoi等人，1990）、水稻（平冢（Hiratsuka）等人，1989）和地钱（Marchantia polymorpha）（Ohyama等，1986年）。每一个叶绿体基因组都编码一个相同的核糖互补体-
Revised Manuscript Received April 27, 1992 abstract: A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector Xgtl 1. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an Mr 19 641 precursor to an M, 14 420 mature protein which is homologous to Escherichia coli ribosomal protein L27. One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly (A) 29 tail, A/3-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein. The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corre-sponding portion of yeast MRP7. The carboxyl end of tobacco L27 has an unusual cysteine-rich sequence (CFCCC) of unknown function. Northern blot analysis revealed a single band of mRNA corresponding in size (0.85-0.90 kb) to full-length L27 cDNAs. Two cDNAs for tobacco ribosomal protein L27 were identical in coding sequence, but differed in 3'-noncoding sequence. Hybridization of L27 cDNA probes to restriction enzyme digests of tobacco genomic DNA and to cloned genomic fragments provided additional evidence suggesting that more than one gene exists for tobacco L27.Ribosomes are protein-synthesizing organelles found in all cells. Thestructure and function of bacterial ribosomes have been intensively studied (Lindahl& Zengel, 1986; Gourse et al., 1986). Recent studies have begun to reveal details about the structure and function of eucaryotic ribosomes (Planta et al., 1986; Warner et al „1986; Wool et al., 1990; Amaldi et al., 1989; Subramanian et al., 1990). In contrast to bacteria, plant cells maintain multiple, distinctly different types of ri-bosomes in the cytoplasm, chloroplast, and mitochondrion (Boynton et al., 1980). These three protein-synthesizing systems differ in the mass and density of their ribosomal subunits, the number of their component proteins, and the size and degree of sequence homology of the rRNA of large and small ribosomal subunits (Boynton et al., 1980; Capel & Bourque, 1982; Bonham-Smith & Bourque, 1990). The genes encoding chloroplast ribosomal proteins are distributed between the chloroplast and nuclear genomes. Twenty-one ribosomal protein genes occur in chloroplast ge-nomes of tobacco (Shinozaki et al., 1986; Yokoi et al., 1990), rice (Hiratsuka et al., 1989), and the liverwort Marchantia polymorpha (Ohyama et al., 1986). Each of these chloroplast genomescodes for an identical complement of riboso-