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首页> 外文期刊>Molecular and Cellular Biology >petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.
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petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

机译：莱茵衣藻叶绿体中的petD mRNA成熟：5'核酸内切加工的作用。

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Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.
机译：在高级植物叶绿体基因的表达过程中会发生初级转录物的复杂处理。在莱茵衣藻中，大多数叶绿体基因似乎拥有自己的启动子，而不是作为多顺反子操纵子的一部分转录。通过生成特定的缺失突变体，我们表明，编码细胞色素b6 / f复合物的亚基IV的petD具有在petD启动子缺失突变体中积累单顺反子petD mRNA所需的RNA处理位点;在此类突变体中，petD的转录源自上游petA启动子。在petD启动子上起始的转录本的5'端也可能是通过加工产生的，因为单顺反子petD mRNA的5'端在野生型菌株中与在petD启动子突变体中是相同的。通过将petD-uidA融合基因插入叶绿体基因组（uidA是编码β-葡萄糖醛酸苷酶的大肠杆菌基因），进一步检查了加工位点的位置和功能。当将无启动子的petD-uidA融合基因插入petA的下游时，单顺反子uidA转录物蓄积，这显然是在petA启动子处启动的，并在与petD mRNA 5'端精确对应的位点进行加工。当仅包含相对于成熟mRNA 5'端在+25下游的序列的构建体插入同一位点时，双顺反子petA-uidA转录物积累，但未检测到单顺反子uidA转录物，表明加工位点至少部分位于在-1至+25范围内。在仅积累双顺反子petA-uidA转录本的转化子中未检测到β-葡萄糖醛酸苷酶活性，这表明翻译起始需要5'非翻译区的前25 bp。这种翻译缺陷的一种解释是衣藻叶绿体不能翻译某些双顺反子信息的第二编码区。

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