Yan Shannon, Wen Jin-Der, Bustamante Carlos, Tinoco Ignacio
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
Cell. 2015 Feb 26;160(5):870-881. doi: 10.1016/j.cell.2015.02.003. Epub 2015 Feb 19.
Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.
程序性核糖体移码可从单个转录本产生不同的蛋白质。-1移码发生在含有滑序列AAAAAAG和周边mRNA结构屏障的大肠杆菌dnaX mRNA上。在这里,我们揭示了移码过程的隐藏方面,包括其在mRNA上的确切位置及其在翻译周期中的时间。翻译产物的质谱分析表明,核糖体不是从一个特定密码子而是沿着滑序列的各种密码子进入-1框架,并且不仅滑动-1个核苷酸,还滑动-4或+2个核苷酸。单核糖体翻译轨迹检测到核糖体-mRNA在滑序列上位移的独特密码子尺度波动,代表移码过程中的多次核糖体易位尝试。侧翼mRNA结构屏障机械刺激核糖体进行来回易位偏移,广泛探索阅读框。两个实验都揭示了突变滑序列周围的翻译中止,表明对新采用的密码子位置碱基配对的后续保真度检查导致翻译恢复或提前终止。