Dinesh-Kumar S P, Miller W A
Plant Pathology Department, Iowa State University, Ames 50011-1020.
Plant Cell. 1993 Jun;5(6):679-92. doi: 10.1105/tpc.5.6.679.
The signals that control initiation of translation in plants are not well understood. To dissect some of these signals, we used a plant viral mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype, the coat protein (CP) and overlapping 17K open reading frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relative to the AUG codons in efficiency of translation initiation were investigated by translation of sgRNA1 mutants in a cell-free extract and by expression of a reporter gene from mutant sgRNA1 leaders in protoplasts. The effects of mutations that disrupted and restored secondary structure encompassing the CP AUG independently of, and in combination with, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary structure. Secondary structure had an overall inhibitory effect on translation of both ORFs. In general, the "Kozak rules" of start codon preference predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To explain this observation, we propose a new model in which pausing of the ribosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structure in controlling translation initiation should be of value for understanding expression of any plant gene and in the design of artificial constructs.
植物中控制翻译起始的信号尚未得到充分理解。为了剖析其中一些信号，我们使用了一种植物病毒mRNA，其蛋白质合成起始于两个移码起始密码子。在大麦黄矮病毒-PAV血清型的大亚基因组RNA（sgRNA1）上，外壳蛋白（CP）和重叠的17K开放阅读框（ORF）分别从第一个和第二个AUG密码子开始翻译。通过在无细胞提取物中翻译sgRNA1突变体以及在原生质体中从突变sgRNA1前导序列表达报告基因，研究了相对于AUG密码子-3和+4位置的碱基在翻译起始效率中的作用。还研究了独立于以及与-3和+4碱基变化相结合破坏和恢复包含CP AUG的二级结构的突变的影响。用结构敏感核酸酶对sgRNA1前导序列的5'端进行部分消化得到的产物与预测的二级结构一致。二级结构对两个ORF的翻译总体上具有抑制作用。一般来说，起始密码子偏好的“科扎克规则”在决定起始密码子选择中占主导地位。出乎意料的是，对于给定的CP AUG序列背景，降低下游17K AUG起始的变化也会降低CP AUG的起始。为了解释这一观察结果，我们提出了一个新模型，其中核糖体在第二个AUG处的暂停允许在第一个AUG处增加起始。这种对一级和二级结构在控制翻译起始中作用的详细分析对于理解任何植物基因的表达以及人工构建体的设计都应该是有价值的。
