Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Nucleic Acids Res. 2021 Dec 16;49(22):12943-12954. doi: 10.1093/nar/gkab1172.
Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of -1PRF is coordinated by a 'slippery' sequence in the transcript, an adjacent RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop reduce the efficiency of -1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of -1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.
核糖体程序性移码（PRF）是一种翻译重编码机制，可使单个转录本合成多种多肽。在甲型病毒结构多蛋白的翻译过程中，通过转录本中的“滑动”序列、相邻的 RNA 茎环和新生多肽链的构象转变来协调-1PRF 的效率。为了表征这些效应物中的每一种，我们通过深度突变扫描测量了 4530 个突变对-1PRF 的影响。虽然滑动位点和茎环内的大多数突变会降低-1PRF 的效率，但滑动位点上游突变的影响则更为多样。我们确定了几个区域，其中新生多肽的氨基酸序列修饰会影响-1PRF 的效率。多蛋白生物发生的分子动力学模拟表明，这些突变的影响主要源于它们对由易位子介导的新生多肽链共翻译折叠产生的机械力的影响。最后，我们提供的证据表明，共翻译折叠和-1PRF 之间的偶联取决于滑动位点上游的翻译动力学。这些发现表明了-1PRF 如何通过转录本和新生链内的特征进行协调。
