The frequency of translational misreading errors in E. coli is largely determined by tRNA competition
The frequency of translational misreading errors in E. coli is largely determined by tRNA competition
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DOI：
10.1261/rna.294907
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发表时间：
2007-01-01
期刊：
影响因子：
4.5
通讯作者：
Farabaugh, Philip J.
中科院分区：
文献类型：
--
作者：
Kramer, Emily B.;Farabaugh, Philip J.
蛋白质合成过程中错义错误率（误读）的估计值从每个密码子 10(-3) 到 10(-4) 不等。报告这些速率的实验使用多种方法和报告系统测量了几种不同的错误。报告率的变化可能反映了测试错误率或报告系统灵敏度的实际差异。为了更准确地了解错误率的范围，我们开发了一个系统来量化大肠杆菌中定义的密码子的每个可能的误读错误的频率。该系统在萤火虫荧光素酶的活性位点使用必需的赖氨酸。 Lys529 的突变导致活性降低高达 1600 倍，但表型因氨基酸而异。我们假设一些突变基因的残余活性可能是由于 tRNA(UUU)(Lys)（赖氨酸密码子 AAA 和 AAG 的同源 tRNA）对突变密码子的误读造成的。我们的数据验证了这一假设，并揭示了近同源密码子的相对错义错误率的详细信息。事实上，大肠杆菌的错误率差异很大。变异的来源之一是同源 tRNA 对突变密码子的竞争效应；较高的错误频率是由于低丰度 tRNA 的竞争较低所致。我们还使用该系统研究了已知影响错误率的核糖体蛋白突变的影响以及引起错误的抗生素的影响，发现它们仅影响近邻密码子子集的误读，并且它们的影响可能没有以前想象的那么普遍。
Estimates of missense error rates (misreading) during protein synthesis vary from 10(-3) to 10(-4) per codon. The experiments reporting these rates have measured several distinct errors using several methods and reporter systems. Variation in reported rates may reflect real differences in rates among the errors tested or in sensitivity of the reporter systems. To develop a more accurate understanding of the range of error rates, we developed a system to quantify the frequency of every possible misreading error at a defined codon in Escherichia coli. This system uses an essential lysine in the active site of firefly luciferase. Mutations in Lys529 result in up to a 1600-fold reduction in activity, but the phenotype varies with amino acid. We hypothesized that residual activity of some of the mutant genes might result from misreading of the mutant codons by tRNA(UUU)(Lys), the cognate tRNA for the lysine codons, AAA and AAG. Our data validate this hypothesis and reveal details about relative missense error rates of near-cognate codons. The error rates in E. coli do, in fact, vary widely. One source of variation is the effect of competition by cognate tRNAs for the mutant codons; higher error frequencies result from lower competition from low-abundance tRNAs. We also used the system to study the effect of ribosomal protein mutations known to affect error rates and the effect of error-inducing antibiotics, finding that they affect misreading on only a subset of near-cognate codons and that their effect may be less general than previously thought.