Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
Bioorg Med Chem. 2013 Mar 1;21(5):1088-96. doi: 10.1016/j.bmc.2013.01.002. Epub 2013 Jan 9.
Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions+β-alanyl-tRNA(CUA), the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted β-amino acids, and direct evidence for β-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted β-amino acids, including α-methyl-β-alanine, β,β-dimethyl-β-alanine, β-phenylalanine, and β-(p-bromophenyl)alanine. The β-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of β-alanine and representative α-l-amino acids. The isolated proteins containing the modified β-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the β-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of β,β-dimethyl-β-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the β-amino acid.
核糖体中 23S rRNA 的三个区域发生修饰，这些修饰均靠近核糖体肽基转移酶中心 (PTC)，之前曾被用作体外蛋白质生物合成实验的 S-30 制剂的来源。当在含有 UAG 密码子的 mRNA 存在下使用时，这些修饰的核糖体在预定位置+β-丙氨酸-tRNA(CUA) 处产生了更高水平的全长蛋白质，从而抑制了 UAG 密码子。在本研究中，通过使用取代的β-氨基酸扩展了早期的结果，并提供了β-氨基酸掺入的直接证据。目前，有五个具有修饰核糖体的克隆用于使用四种取代的β-氨基酸的实验，包括α-甲基-β-丙氨酸、β,β-二甲基-β-丙氨酸、β-苯丙氨酸和β-(对溴苯基)丙氨酸。β-氨基酸被掺入到大肠杆菌二氢叶酸还原酶 (DHFR) 的三个不同位置（10、18 和 49），并比较了它们对 UAG 密码子的抑制效率与β-丙氨酸和代表性的α-氨基酸的抑制效率。含有修饰β-氨基酸的分离蛋白进行了蛋白水解消化，所得片段通过质谱法进行了表征，证明了β-氨基酸已被掺入 DHFR 中，并且仅存在于预期的肽片段中。DHFR 中第 17 位含有谷氨酸，之前已经证明 Glu-C 内切蛋白酶可以在第 17 和 18 位氨基酸残基之间水解 DHFR。β,β-二甲基-β-丙氨酸被掺入 DHFR 的 18 位阻止了这种切割，为β-氨基酸的掺入位置提供了进一步的证据。
