RRM
2013 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Thg1 and Thg1-like proteins (TLPs) are found in Eukarya, Archaea, and Bacteria. Thg1 is a part of a novel class of enzymes that catalyzes 3&prime to 5&prime nucleotide addition to tRNAHis as the final essential maturation step of this tRNA. Other Thg1 family members utilize 3&prime - 5&prime polymerase activity to repair the 5&prime -ends of tRNA during 5&prime -tRNA editing, however, additional unidentified roles for these enzymes in biology are likely.
The crystal structure of human Thg1 (hThg1) complexed with two molecules of GTP confirmed that hThg1 utilizes a two-metal ion mechanism for G-1 addition and provided evidence for the binding site for the activating NTP; however, it did not provide any information on Thg1 interaction with tRNAHis . Previous work identified critical residues for G-1 addition, including several residues (D68, H152 and N198) that appear to play a role in tRNA binding and/or recognition, according to biochemical assays. However, the molecular basis for the involvement of these residues and others in binding to tRNAHis is not known.
Nuclease footprinting is being used to provide approximate regions of tRNAHis that may be involved in hThg1 binding. At present, nuclease digestion conditions have been optimized. Preliminary nuclease footprinting experiments suggest that there are some protected regions of tRNAHis in the presence of hThg1. However, the use of a nuclease-based method to determine RNA-protein contacts has limitations including abrogation of hThg1 binding to tRNAHis. Furthermore, bulky nucleases are more sensitive to steric hindrance, especially in the presence of protein. Additionally, ribonuclease cleavage could alter the structure of the RNA and cause secondary cleavages. Therefore, additional complementary approaches will also be employed to investigate and strengthen conclusions from these data.
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