Hao Yumeng, Hulscher Ryan M, Zinshteyn Boris, Woodson Sarah A
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA.
bioRxiv. 2024 Jan 10:2024.01.10.574868. doi: 10.1101/2024.01.10.574868.
The production of new ribosomes requires proper folding of the rRNA and the addition of more than 50 ribosomal proteins. The structures of some assembly intermediates have been determined by cryo-electron microscopy, yet these structures do not provide information on the folding dynamics of the rRNA. To visualize the changes in rRNA structure during ribosome assembly in cells, transcripts were pulse-labeled with 4-thiouridine and the structure of newly made rRNA probed at various times by dimethyl sulfate modification and mutational profiling sequencing (4U-DMS-MaPseq). The in-cell DMS modification patterns revealed that many long-range rRNA tertiary interactions and protein binding sites through the 16S and 23S rRNA remain partially unfolded 1.5 min after transcription. By contrast, the active sites were continually shielded from DMS modification, suggesting that these critical regions are guarded by cellular factors throughout assembly. Later, bases near the peptidyl tRNA site exhibited specific rearrangements consistent with the binding and release of assembly factors. Time-dependent structure-probing in cells suggests that many tertiary interactions throughout the new ribosomal subunits remain mobile or unfolded until the late stages of subunit maturation.
新核糖体的产生需要rRNA的正确折叠以及50多种核糖体蛋白的添加。一些组装中间体的结构已通过冷冻电子显微镜确定，但这些结构并未提供有关rRNA折叠动力学的信息。为了观察细胞中核糖体组装过程中rRNA结构的变化，转录本用4-硫尿苷进行脉冲标记，并在不同时间通过硫酸二甲酯修饰和突变分析测序（4U-DMS-MaPseq）探测新合成的rRNA的结构。细胞内DMS修饰模式显示，转录1.5分钟后，16S和23S rRNA中的许多长程rRNA三级相互作用和蛋白质结合位点仍部分未折叠。相比之下，活性位点持续免受DMS修饰，这表明在整个组装过程中这些关键区域受到细胞因子的保护。后来，肽基tRNA位点附近的碱基表现出与组装因子的结合和释放一致的特定重排。细胞内随时间变化的结构探测表明，新核糖体亚基中的许多三级相互作用在亚基成熟后期之前仍保持动态或未折叠状态。
