Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
RNA. 2023 Oct;29(10):1500-1508. doi: 10.1261/rna.079690.123. Epub 2023 Jul 7.
The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.
核糖体是一种大型核糖核蛋白复合物，利用多种复杂的分子相互作用来维持正确的折叠。通过在 16S 或 23S 核糖体 RNA（rRNA）中安装 MS2 标签，已分离出体内组装的核糖体，从而能够在体外研究核糖体的结构和功能。通常将 RNA 标签插入到 23S rRNA 的延伸螺旋 H98 中，因为这种添加不会影响细胞生长或体外核糖体的活性。在这里，我们发现与野生型（WT）50S 亚基相比，在 H98 中插入 MS2 标签的 50S 亚基不稳定。我们确定了导致不稳定的原因是破坏了连接 H1、H94 和 H98 螺旋的 RNA-RNA 三级接触。通过低温电子显微镜（cryo-EM），我们表明这种相互作用会因 MS2 标签的添加而被破坏，并且可以通过在延伸的 H98 螺旋中插入单个腺苷来恢复。这项工作建立了改进 50S 亚基中 MS2 标签的方法，这些标签可维持核糖体的稳定性，并研究了一种复杂的 RNA 三级结构，该结构可能对各种细菌核糖体的稳定性很重要。
