Daniels Kate G, Radrizzani Sofia, Hurst Laurence D
Milner Centre for Evolution, Department of Life Sciences, University of Bath, Bath, BA2 7AY, UK.
NAR Genom Bioinform. 2025 Jun 19;7(2):lqaf086. doi: 10.1093/nargab/lqaf086. eCollection 2025 Jun.
In bacteria, high A and low G content of the 5' end of the coding sequence (CDS) promotes low RNA stability, facilitating ribosomal initiation and subsequently a high protein to transcript ratio. Additionally, 5' NGG codons are suppressive owing to peptidyl-tRNA drop off. It was, therefore, surprising that the first large-scale transgene experiment to interrogate the 5' effect by codon randomization found the NGG, G-rich codon AGG to be the most associated with high transgene output. Why is this? We show that this is not replicated in other large transgene datasets, where AGG and NGG are associated with low efficiency. More generally, there is limited agreement between the first experiment and others. This we find to be a consequence of non-random construct design. In constructs of the first experiment, AGG disproportionately occurs with non-AGG codons associated with low stability and high protein output, making AGG's association with high output an artefact. While translationally non-optimal codons like AGG are conjectured to slow ribosomes for orderly initiation, we find that in the less biased constructs high, not low, translational adaptation in the first 10 codons is (weakly) predictive of higher translational efficiency. These results have implications for both transgene and experimental design.
在细菌中，编码序列（CDS）5'端的高A含量和低G含量会降低RNA稳定性，促进核糖体起始，进而导致高蛋白质与转录本比率。此外，5' NGG密码子由于肽基-tRNA脱落而具有抑制作用。因此，令人惊讶的是，首个通过密码子随机化来探究5'端效应的大规模转基因实验发现，富含G的密码子AGG与高转基因表达量最为相关。这是为什么呢？我们发现，在其他大规模转基因数据集中并未出现这种情况，在这些数据集中，AGG和NGG与低效率相关。更普遍地说，首个实验与其他实验之间的一致性有限。我们发现这是由于非随机构建设计导致的。在首个实验的构建体中，AGG不成比例地与低稳定性和高蛋白质输出相关的非AGG密码子同时出现，使得AGG与高输出之间的关联成为一种假象。虽然像AGG这样翻译上非最优的密码子被推测会使核糖体减速以实现有序起始，但我们发现，在偏差较小的构建体中，前10个密码子的高（而非低）翻译适应性（微弱地）预示着更高的翻译效率。这些结果对转基因和实验设计都有影响。
