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Information × Registration Number 0225U001090, (0119U103634) , R & D reports Title Molecular mechanisms for quality control of translation with non-proteinogenic amino acids participation popup.stage_title Клонування і експресія в E.coli аланіл-тРНК синтетаз Enterococcus faecalis і людини та вивчення їх редагувальної активності відносно D-аланіну. Head Tukаlo Mykhаilo A., Доктор біологічних наук Registration Date 24-01-2025 Organization Institute of Molecular Biology and Genetics of NAS of Ukraine popup.description1 The goal of this project is to study the structural and conformational determinants of tRNAs, human leucyl-tRNA synthetase and alanyl-tRNA synthetases from various species that exclude the participation of non-proteinogenic amino acids such as norleucine, norvaline, homocysteine and D-alanine, in protein biosynthesis. The mechanisms of stereospecificity control in translation of genetic information have not yet been elucidated. Therefore, the ability of AlaRS and ProRS from different species (E. coli, Enterococcus faecalis and Homo sapiens) to edit D-amino acids will be studied. Additionally, separate trans-editing factor AlaX will be examined. The comprehensive approach, combining genetic engineering, biochemical, biophysical and molecular dynamics methods and quantum chemical calculations, will be applied. The study of the influence of incorporation of norvaline, norleucine and homocysteine into proteins on the growth of normal and cancer cells will be performed. The results of our investigations will be used for the search of new antibiotics against human pathogens and new drugs against cancer. Additionally, they also provide the basis for the creation of new orthologous tRNA-ARSase pairs for synthetic biology. popup.description2  During this stage, the conditions for the expression of alanine-tRNA synthetase from Enterococcus faecalis in E. coli cells and its purification were optimized. The expression and purification conditions for human AlaRS were also optimized. The purification conditions for the trans-editing factor AlaX from Thermus thermophilus were developed. The kinetic parameters of the activation of cognate and non-cognate amino acids by alanine-tRNA synthetase from T. thermophilus and its mutant form AlaRS (C666A) from E. coli were studied. For AlaRS from T. thermophilus, the pyrophosphate exchange reaction was also investigated. Additionally, the kinetic parameters of the aminoacylation reaction for TyrRS from T. thermophilus were determined. Product Description popup.authors Hudzera Olha Y. Kovalenko Oksana P. Kryklyvyi Ivan A. Raievskyi Oleksii V. Rybak Mariia Yu. Skydanovych Oleksandra I. Yaremchuk Hanna D. popup.nrat_date 2025-01-24 Close