Aval Sepideh Fakhretaha, Seffouh Amal, Moon Kyung-Mee, Foster Leonard J, Ortega Joaquin, Fredrick Kurt
Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
RNA. 2025 Mar 18;31(4):585-599. doi: 10.1261/rna.080335.124.
The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, form contacts with the SRL that are critical for GTP hydrolysis and factor function. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in Isolated ΔSRL particles were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We find that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this activity is blocked in the absence of the SRL.
肌动蛋白-蓖麻毒素环（SRL）是核糖体RNA（rRNA）中最保守的片段之一。翻译型GTP酶（trGTPases），如EF-G、EF-Tu和IF2，与SRL形成接触，这对GTP水解和因子功能至关重要。先前的研究表明，缺乏SRL的23S rRNA的表达在大肠杆菌中赋予显性致死表型。发现分离的ΔSRL颗粒不仅在蛋白质合成中无活性，而且组装不完全。特别是，包括肽基转移酶中心的亚基的第4块仍然未折叠。在这里，我们探讨这种组装缺陷的基础。我们发现从ΔSRL亚基中提取的23S rRNA可以有效地重新组装成50S亚基，并且这些重新组装的ΔSRL颗粒表现出完全的肽基转移酶活性。我们还使用冷冻电镜和蛋白质组学方法进一步表征从细胞中纯化的ΔSRL颗粒。这些颗粒缺乏第4块rRNA和r蛋白的密度，这与早期的化学探测数据一致。用大亚基的过量总r蛋白（TP50）孵育这些颗粒未能恢复大量的肽基转移酶活性。有趣的是，对照颗粒和突变颗粒的蛋白质组分析表明，在ΔSRL情况下，多种组装因子的比例过高。我们提出一种或多种GTP酶通常起到释放组装因子的作用，并且在没有SRL的情况下这种活性被阻断。
