Sakamoto W, Sturm N R, Kindle K L, Stern D B
Boyce Thompson Institute for Plant Research at Cornell University, Ithaca, New York 14853.
Mol Cell Biol. 1994 Sep;14(9):6180-6. doi: 10.1128/mcb.14.9.6180-6186.1994.
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.
高等植物叶绿体基因表达过程中会发生初级转录本的复杂加工。在莱茵衣藻中，大多数叶绿体基因似乎都有自己的启动子，而不是作为多顺反子操纵子的一部分进行转录。通过构建特定的缺失突变体，我们发现编码细胞色素b6/f复合体亚基IV的petD有一个RNA加工位点，该位点是petD启动子缺失突变体中单个顺反子petD mRNA积累所必需的；在这类突变体中，petD的转录起始于上游的petA启动子。在petD启动子处起始的转录本的5'端可能也是通过加工产生的，因为单个顺反子petD mRNA的5'端在野生型菌株和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是大肠杆菌中编码β-葡萄糖醛酸酶的基因），进一步研究了加工位点的位置和功能。当一个无启动子的petD-uidA融合基因插入到petA下游时，积累了一个单个顺反子的uidA转录本，该转录本显然起始于petA启动子，并在一个与petD mRNA 5'端精确对应的位点进行加工。当一个仅包含相对于成熟mRNA 5'端+25下游序列的构建体插入到同一位点时，积累了一个双顺反子petA-uidA转录本，但未检测到单个顺反子的uidA转录本，这表明加工位点至少部分位于-1至+25区域内。在仅积累双顺反子petA-uidA转录本的转化体中未检测到β-葡萄糖醛酸酶活性，这表明5'非翻译区的前25个碱基对是翻译起始所必需的。对这种翻译缺陷的一种解释是，莱茵衣藻叶绿体无法翻译某些双顺反子信息的第二个编码区。
