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Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides |
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| Authors: | Takayuki Kanda Kazuyuki Takai Shigeyuki Yokoyama Hiroshi Takaku |
| Institution: | aDepartment of Industrial Chemistry, Faculty of Engineering, Chiba Institute of Technology, 2-17-1, Tsudanuma, Narashino, Chiba 275-0016, Japan;bDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan |
| Abstract: | Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNAAsp blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This `knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons. |
| Keywords: | Cell-free protein synthesis Antisense oligodeoxyribonucleotide tRNA Aminoacylation Escherichia coli |
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