Jungbauer Lisa M, Bakke Courtney K, Cavagnero Silvia
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
J Mol Biol. 2006 Apr 7;357(4):1121-43. doi: 10.1016/j.jmb.2006.01.012. Epub 2006 Jan 30.
This work focuses on the experimental analysis of the time-course of protein expression in a cell-free system, in conjunction with the development of a computational model, denoted as progressive chain buildup (PCB), able to simulate translation kinetics and product formation as a function of starting reactant concentrations. Translation of the gene encoding the apomyoglobin (apoMb) model protein was monitored in an Escherichia coli cell-free system under different experimental conditions. Experimentally observed protein expression yields, product accumulation time-course and expression completion times match with the predictions by the PCB model. This algorithm regards elementary single-residue elongations as apparent second-order events and it accounts for aminoacyl-tRNA regeneration during translation. We have used this computational approach to model full-length protein expression and to explore the kinetic behavior of incomplete chains generated during protein biosynthesis. Most of the observed incomplete chains are non-obligatory dead-end species, in that their formation is not mandatory for full-length protein expression, and that they are unable to convert to the expected final translation product. These truncated polypeptides do not arise from post-translational degradation of full-length protein, but from a distinct subpopulation of chains which expresses intrinsically more slowly than the population leading to full-length product. The PCB model is a valuable tool to predict full-length and incomplete chain populations and formulate experimentally testable hypotheses on their origin. PCB simulations are applicable to E.coli cell-free expression systems (both in batch and dialysis mode) under the control of T7 RNA polymerase and to other environments where transcription and translation can be regarded as kinetically decoupled.
这项工作聚焦于在无细胞系统中对蛋白质表达时间进程的实验分析，同时开发了一种计算模型，称为渐进链积累（PCB），该模型能够模拟翻译动力学以及作为起始反应物浓度函数的产物形成。在不同实验条件下，在大肠杆菌无细胞系统中监测编码脱辅基肌红蛋白（apoMb）模型蛋白的基因的翻译。实验观察到的蛋白质表达产量、产物积累时间进程和表达完成时间与PCB模型的预测结果相符。该算法将基本的单残基延伸视为表观二级事件，并考虑了翻译过程中氨酰-tRNA的再生。我们已使用这种计算方法对全长蛋白质表达进行建模，并探索蛋白质生物合成过程中产生的不完全链的动力学行为。观察到的大多数不完全链是非必需的终产物，因为它们的形成对于全长蛋白质表达并非必需，并且它们无法转化为预期的最终翻译产物。这些截短的多肽并非源自全长蛋白质的翻译后降解，而是源自一个独特的链亚群，该亚群的表达本质上比导致全长产物的群体更慢。PCB模型是预测全长链和不完全链群体并就其起源提出可通过实验验证的假设的宝贵工具。PCB模拟适用于在T7 RNA聚合酶控制下的大肠杆菌无细胞表达系统（批式和透析模式）以及其他转录和翻译可被视为动力学解耦的环境。
