The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved ⁴⁴PNSA⁴⁷ loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.

中文翻译：

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核糖体蛋白 uS12 的突变揭示了其普遍保守的 PNSA 环的新功能

核糖体蛋白 uS12 在生命的所有领域都是保守的。最近，人类 uS12 中的杂合自发突变（对应于大肠杆菌uS12 中普遍保守的⁴⁴ PNSA ⁴⁷环下游的 R49K 突变）被确定为导致核糖体病，突出了 PNSA 环的重要性。为了研究在没有任何野生型等位基因的情况下类似突变的影响，我们对大肠杆菌中的rpsL基因（编码 uS12）进行了突变。与其病理学（在人类中）一致，我们无法在大肠杆菌中产生 R49K 突变在没有支持质粒的情况下。然而，我们能够在其附近产生 L48K 突变。L48K 突变导致该菌株出现冷敏感表型和核糖体生物发生缺陷。我们表明 L48K 突变会影响起始和延伸的步骤。此外，L48K 突变与 RRF 和 Pth 的遗传相互作用表明 PNSA 环在核糖体循环中的新作用。我们的研究揭示了 uS12 中 PNSA 环的新功能，迄今为止已在翻译延伸的背景下对其进行了研究。