Studying Mutations in the Trm10 Methyltransferase to Reveal the Roles of Conserved Residues and Domains in Substrate Specificity and Catalytic Activity
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Date
2021-05
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The Ohio State University
Abstract
tRNA modifications are a pivotal aspect of biology observed across all domains of life. These modifications rely on the presence of enzymes to induce the addition or deletion of groups on a tRNA. One of these enzymes is Trm10, which exists as three different homologs within the human body: denoted TRMT10A, TRMT10B, and TRMT10C. These proteins methylate at the N-1 position of nucleic acids at the 9th position of the tRNA, such that TRMT10A methylates a guanine to make N-1 methylguanosine (at the 9th position is denoted m1G9), TRMT10B methylates an adenine to make N-1 methyladenosine (at the 9th position is denoted m1A9), and TRMT10C performs both m1G9 and m1A9. In recent years, studies have demonstrated the importance of the SpoU Trm-D core of the protein (SPOUT core) in relation to this activity, but relatively little is known about the significant number of upstream and downstream residues on the protein, which comprise its N and C-terminal domains. These protein domains have little sequence conservation across species and domains of life, and their role in Trm10 activity remains unknown.
This project seeks to elucidate whether the presence of different upstream and downstream sequences would alter the activity of the SPOUT core of each type of TRMT10 by creating chimeric proteins where sequences from TRMT10A and B are mixed together. This way, terminal domains of TRMT10A are connected to a TRMT10B core and vice versa. Upon completion of this work, I have demonstrated that a construct with a TRMT10A core and TRMT10B upstream and downstream fragments (denoted BAB) does not show any activity, while all other TRMT10A core chimera retain m1G9 activity. On the other hand, no TRMT10B chimeric proteins displayed any activity in the assays I performed. These results suggest the possibility that there is a role played in having at least one of the TRMT10A terminal domains present with the TRMT10A core, but this same phenomena does not exist with the TRMT10B core.
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Keywords
Biochemistry, tRNA Modification, Trm10, Chimera