Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center
Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center
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DOI：
10.1016/j.bmc.2013.01.002
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发表时间：
2013-03-01
影响因子：
3.5
通讯作者：
Hecht, Sidney M.
中科院分区：
文献类型：
--
作者：
Maini, Rumit;Nguyen, Dan T.;Hecht, Sidney M.
在23 S rRNA的三个区域中含有修饰的核糖体，所有这些区域都靠近核糖体肽基转移酶中心（PTC），以前被用作体外蛋白质生物合成实验的S-30制剂的来源。当在预定位置含有UAG密码子的mRNA + β-丙氨酰-tRNA（CUA）存在下使用时，修饰的核糖体通过UAG密码子抑制产生增强水平的全长蛋白质。在本研究中，这些早期的结果已经通过使用取代的β-氨基酸得到了扩展，并提供了β-氨基酸掺入的直接证据。目前，五个具有修饰的核糖体的克隆用于采用四种取代的β-氨基酸的实验中，所述取代的β-氨基酸包括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。将β-氨基酸掺入大肠杆菌二氢叶酸还原酶（DHFR）的三个不同位置（10、18和49），并将它们抑制UAG密码子的效率与β-丙氨酸和代表性α-L-氨基酸的效率进行比较。将含有修饰的β-氨基酸的分离的蛋白质进行蛋白水解消化，并通过质谱法表征衍生的片段，确定β-氨基酸已掺入DHFR中，并且它们仅存在于预期的肽片段中。DHFR在17位含有谷氨酸，并且先前已经显示Glu-C内切蛋白酶可以水解氨基酸残基17和18之间的DHFR。将β，β-二甲基-β-丙氨酸掺入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。(C)2013爱思唯尔有限公司保留所有权利。
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 + beta-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 beta-amino acids, and direct evidence for beta-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted beta-amino acids, including alpha-methyl-beta-alanine, beta,beta-dimethyl-beta-alanine, beta-phenylalanine, and beta-(p-bromophenyl)alanine. The beta-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 beta-alanine and representative alpha-L-amino acids. The isolated proteins containing the modified beta-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the beta-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 beta,beta-dimethyl-beta-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the beta-amino acid. (C) 2013 Elsevier Ltd. All rights reserved.