Eukaryotic translation initiation involves preinitiation ribosomal complex 5′-to-3′ directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context.

中文翻译：

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人类 mRNA 中注释起始密码子下游的翻译起始与 Kozak 上下文共同进化。

真核翻译起始包括预起始核糖体复合物 5' 到 3' 定向探测 mRNA 以寻找适合起始蛋白质合成的密码子。密码子作为起始的识别取决于密码子身份及其直接核苷酸上下文，称为 Kozak 上下文。当环境较弱（即非最佳）时，会发生泄漏扫描，在此期间，一小部分核糖体继续进行 mRNA 探测。我们探讨了注释为蛋白质编码序列开始的 AUG 密码子上下文与下一个 AUG 密码子出现之间的关系。我们发现弱起始下游的 AUG 密码子比强起始下游更频繁地出现在同一帧中。我们建议对弱起始密码子下游的框内 AUG 进行进化选择是由减少浪费的框外产物合成的优势以及从某些 mRNA 产生多种蛋白质型的优势驱动的。我们使用核糖体分析数据确认了弱起始密码子下游的翻译起始。我们还使用报告构建体测试了 10 个特定人类基因中替代起始密码子的翻译。在所有测试案例中，下游起​​始密码子的起始比带注释的起始密码子更有效率。在大多数情况下，Kozak 上下文的优化并没有完全取消下游启动，在具体的例子中 我们使用核糖体分析数据确认了弱起始密码子下游的翻译起始。我们还使用报告构建体测试了 10 个特定人类基因中替代起始密码子的翻译。在所有测试案例中，下游起​​始密码子的起始比带注释的起始密码子更有效率。在大多数情况下，Kozak 上下文的优化并没有完全取消下游启动，在具体的例子中 我们使用核糖体分析数据确认了弱起始密码子下游的翻译起始。我们还使用报告构建体测试了 10 个特定人类基因中替代起始密码子的翻译。在所有测试案例中，下游起​​始密码子的起始比带注释的起始密码子更有效率。在大多数情况下，Kozak 上下文的优化并没有完全取消下游启动，在具体的例子中CMPK1 mRNA，优化的开始仍然无效。总的来说，我们的工作揭示了以前未表征的影响蛋白质编码基因进化的力量，并指出翻译起始的多样性和影响起始密码子选择的序列特征的存在，而不是 Kozak 上下文。