Characterization of Alanyl-tRNA Synthetase Quality Control in Bacillus subtilis

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The Ohio State University

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Translation is an essential cellular process, resulting in the synthesis of proteins (1). While translation is critical for the cell, it is comparatively error-prone relative to other essential cellular processes. In translation, errors occur approximately once per 10^4 codons. In comparison, the error rate in DNA replication is approximately one error per 10^8 nucleotides (1). It is important to understand how these errors in translation affect the cell. Aminoacyl-tRNA synthetases (aaRSs) are critical enzymes in translation which ligate tRNA to their cognate amino acids. Due to their ability to edit tRNA mischarged with non-cognate amino acids through pre- and post-transfer editing, aaRSs have a role in prevention of errors in translation (1). A lack of aaRS quality control has been shown to be detrimental to the cell (2, 3). While aaRS quality control can be important for the cell, it has been observed that certain aaRSs can mischarge tRNA with non-cognate amino acids. Alanyl-tRNA synthetase (AlaRS) from Streptococcus pneumoniae, a Gram-positive organism, mischarges tRNAAla with serine. Ser-tRNAAla has been shown to be used by MurM, an enzyme involved in peptidoglycan crosslinking in S. pneumoniae (6). To further study AlaRS quality control in Gram-positive organisms, an AlaRS editing deficient Bacillus subtilis strain was made. B. subtilis alaS, which encodes for AlaRS, was cloned into pET28a, and mutagenized to encode C668A AlaRS (8). B. subtilis wild-type (WT) and C668A AlaRS were expressed and purified for characterization in vitro to confirm that the C668A mutation diminished AlaRS editing activity. WT and C668A AlaRS were able to charge tRNAAla with alanine. However, C668A AlaRS was able to mischarge tRNAAla with serine, and had a diminished ability to edit Ser-tRNAAla compared to WT AlaRS. This suggests that C668 is an essential residue for AlaRS editing activity. Lastly, there was no significant difference in growth between the WT and AlaRS editing deficient B. subtilis strains in Luria Broth (LB) or M9 minimal media, which suggests that AlaRS quality control is not required for normal growth in these conditions.



aminoacyl-tRNA synthetase, Alanyl-tRNA Synthetase, Bacillus subtilis, protein synthesis