A mutation in the ribosomal protein uS12 reveals novel functions of its universally conserved PNSA loop

• Published in: Molecular microbiology 2021-06, Vol.115 (6), p.1292-1308
• Main Authors: Datta, Madhurima, Pillai, Maalavika, Modak, Mamata Jayant, Liiv, Aivar, Khaja, Faisal Tarique, Hussain, Tanweer, Remme, Jaanus, Varshney, Umesh
• Format: Article
• Language: English
• Subjects: E coli; Elongation; Evolution; Mutation; peptidyl‐tRNA hydrolase; Phenotypes; Proteins; ribosomal protein; ribosomal subunit; ribosome; RpsL gene; Translation elongation; translation fidelity; Yeast
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.14675

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. The PNSA loop in the ribosomal protein uS12 is universally conserved and has been studied in the context of translation elongation. Using Escherichia coli, we show additional roles the PNSA loop plays in ribosome biogenesis, ribosome association, and the fidelity of translation initiation. Also, our studies reveal genetic interactions between the PNSA loop and RRF. In addition, IF3 initiation from AUU codon may be linked to its (IF3) role in mitigating initiation from non‐AUG codons.