BACKGROUND Single nucleotide substitutions in protein-coding genes can be divided into synonymous (S), with little fitness effect, and non-synonymous (N) ones that alter amino acids and thus generally have a greater effect. Most of the N substitutions are affected by purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases potentially could alleviate the deleterious effect of single substitutions, making them subject to positive selection. To elucidate the effects of selection on double substitutions in all codons, it is critical to differentiate selection from mutational biases. RESULTS We addressed the evolutionary regimes of within-codon double substitutions in 37 groups of closely related prokaryotic genomes from diverse phyla by comparing the fractions of double substitutions within codons to those of the equivalent double S substitutions in adjacent codons. Under the assumption that substitutions occur one at a time, all within-codon double substitutions can be represented as "ancestral-intermediate-final" sequences (where "intermediate" refers to the first single substitution and "final" refers to the second substitution) and can be partitioned into four classes: (1) SS, S intermediate-S final; (2) SN, S intermediate-N final; (3) NS, N intermediate-S final; and (4) NN, N intermediate-N final. We found that the selective pressure on the second substitution markedly differs among these classes of double substitutions. Analogous to single S (synonymous) substitutions, SS double substitutions evolve neutrally, whereas analogous to single N (non-synonymous) substitutions, SN double substitutions are subject to purifying selection. In contrast, NS show positive selection on the second step because the original amino acid is recovered. The NN double substitutions are heterogeneous and can be subject to either purifying or positive selection, or evolve neutrally, depending on the amino acid similarity between the final or intermediate and the ancestral states. CONCLUSIONS The results of the present, comprehensive analysis of the evolutionary landscape of within-codon double substitutions reaffirm the largely conservative regime of protein evolution. However, the second step of a double substitution can be subject to positive selection when the first step is deleterious. Such positive selection can result in frequent crossing of valleys on the fitness landscape.

中文翻译：

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通过密码子内的双重替代跨越健身谷。

背景技术蛋白质编码基因中的单核苷酸取代可分为同义（S），具有很少的适应性作用；和非同义（N），其改变氨基酸并因此通常具有更大的作用。N个替代品中的大多数会受到纯化选择的影响，这些选择将它们排除在不断发展的种群中。但是，附近碱基的其他突变可能会减轻单个取代的有害作用，使其成为阳性选择。为了阐明选择对所有密码子中双取代的影响，将选择与突变偏倚区分开是至关重要的。结果我们通过比较密码子内的双取代部分与相邻密码子中等价的双S取代部分，探讨了37种来自不同门的紧密相关原核基因组中密码子内双取代的进化机制。在一次替换一个的假设下，所有密码子内的双重替换都可以表示为“祖先-中间-最终”序列（其中“中间”是指第一个单个替换，“最终”是指第二个替换）并可以分为四类：（1）SS，S中间-S final；（2）SN，S中间N决赛；（3）NS，N中间体-S final；（4）NN，N个中级，N个决赛。我们发现，在这些类型的双重取代中，对第二次取代的选择压力明显不同。类似于单S（同义）取代，SS双取代是中性进化的，而类似于单N（非同义）取代，SN双取代要经过纯化选择。相反，NS在第二步显示出阳性选择，因为回收了原始氨基酸。NN双取代是异质的，取决于最终状态或中间状态与祖先状态之间的氨基酸相似性，可以进行纯化或阳性选择，也可以进行中性进化。结论目前的结果，密码子内双取代的进化态势的综合分析再次证实了蛋白质进化的保守机制。但是，当第一步有害时，可以对第二步的双重替换进行正面选择。这种积极的选择可能会导致健身景观上的山谷频繁穿越。