Protein Synthesis with Ribosomes Selected for the Incorporation of 尾-Amino Acids

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• 作者：Rumit Maini ; Sandipan Roy Chowdhury ; Larisa M. Dedkova ; Basab Roy ; Sasha M. Daskalova ; Rakesh Paul ; Shengxi Chen ; Sidney M. Hecht
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：June 16, 2015
• 年：2015
• 卷：54
• 期：23
• 页码：3694-3706
• 全文大小：610K
• ISSN：1520-4995

文摘

In an earlier study, 尾³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different 尾-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate 尾-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the 尾³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA_CUA activated with each of four methyl-尾-alanine isomers (1鈥?). The modified ribosomes were found to incorporate each of the four isomeric methyl-尾-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-尾-alanine (尾-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated 尾-tyrosine moiety of 尾³-puromycin. Also conducted were a selection of clones that are responsive to 尾²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-尾-alanine (尾-mAla; 4) into a short 伪-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.