Kozak M
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Mamm Genome. 1996 Aug;7(8):563-74. doi: 10.1007/s003359900171.
This review discusses some rules for assessing the completeness of a cDNA sequence and identifying the start site for translation. Features commonly invoked-such as an ATG codon in a favorable context for initiation, or the presence of an upstream in-frame terminator codon, or the prediction of a signal peptide-like sequence at the amino terminus-have some validity; but examples drawn from the literature illustrate limitations to each of these criteria. The best advice is to inspect a cDNA sequence not only for these positive features but also for the absence of certain negative indicators. Three specific warning signs are discussed and documented: (i) The presence of numerous ATG codons upstream from the presumptive start site for translation often indicates an aberration (sometimes a retained intron) at the 5' end of the cDNA. (ii) Even one strong, upstream, out-of-frame ATG codon poses a problem if the reading frame set by the upstream ATG overlaps the presumptive start of the major open reading frame. Many cDNAs that display this arrangement turn out to be incomplete; that is, the out-of-frame ATG codon is within, rather than upstream from, the protein coding domain. (iii) A very weak context at the putative start site for translation often means that the cDNA lacks the authentic initiator codon. In addition to presenting some criteria that may aid in recognizing incomplete cDNA sequences, the review includes some advice for using in vitro translation systems for the expression of cDNAs. Some unresolved questions about translational regulation are discussed by way of illustrating the importance of verifying mRNA structures before making deductions about translation.
本综述讨论了评估cDNA序列完整性和确定翻译起始位点的一些规则。通常提到的特征，如处于有利于起始的环境中的ATG密码子、上游框内终止密码子的存在，或氨基末端信号肽样序列的预测，都有一定的合理性；但文献中的例子说明了这些标准各自的局限性。最好的建议是，不仅要检查cDNA序列中的这些积极特征，还要检查是否不存在某些负面指标。文中讨论并记录了三个具体的警示信号：(i) 在假定的翻译起始位点上游存在大量ATG密码子，通常表明cDNA的5'端存在异常（有时是保留的内含子）。(ii) 如果上游ATG设定的阅读框与主要开放阅读框的假定起始部分重叠，即使有一个强大的、上游的、框外的ATG密码子也会带来问题。许多呈现这种排列的cDNA结果是不完整的；也就是说，框外ATG密码子在蛋白质编码域内，而不是在其上游。(iii) 假定的翻译起始位点处的环境非常弱，通常意味着cDNA缺乏真正的起始密码子。除了提出一些可能有助于识别不完整cDNA序列的标准外，本综述还包括一些关于使用体外翻译系统表达cDNA的建议。通过说明在对翻译进行推断之前验证mRNA结构的重要性，讨论了一些关于翻译调控的未解决问题。
