Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids.
Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids.
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DOI：
10.1021/acs.biochem.5b00389
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发表时间：
2015-06-16
期刊：
影响因子：
2.9
通讯作者：
Hecht, Sidney M.
中科院分区：
文献类型：
--
作者：
Maini, Rumit;Chowdhury, Sandipan Roy;Dedkova, Larisa M.;Roy, Basab;Daskalova, Sasha M.;Paul, Rakesh;Chen, Shengxi;Hecht, Sidney M.
在早期的研究中，β3-嘌呤霉素用于选择修饰的核糖体，其用于将五种不同的β-氨基酸掺入大肠杆菌二氢叶酸还原酶（DHFR）中。所选的核糖体能够将结构上不同的β-氨基酸掺入DHFR中，尽管使用单一嘌呤霉素来选择单个克隆。在这项研究中，我们检查了用于核糖体选择的β3-嘌呤霉素的结构在多大程度上影响了蛋白质合成过程中修饰的核糖体的区域和立体化学偏好;机制探针是一个单一的抑制剂tRNACUA，由四种甲基-β-丙氨酸异构体中的每一种激活（1-4）。发现修饰的核糖体将四种异构的甲基-β-丙氨酸中的每一种掺入DHFR中，但表现出对3（S）-甲基-β-丙氨酸（β-mAla; 4）的偏好，即，该异构体具有与β3-嘌呤霉素的O-甲基化β-酪氨酸部分相同的区域和立体化学。还进行了对β2-嘌呤霉素有响应的克隆的选择，并证明了这些克隆在蛋白质合成期间的区域和立体化学偏好的逆转。将这些结果纳入23 S rRNA修饰区的结构模型中，该模型包括氢键网络的计算机预测。最后，证明了将3（S）-甲基-β-丙氨酸（β-mAla; 4）掺入hnRNP LL核酸结合结构域的短α-螺旋区域中显著稳定螺旋而不影响其DNA结合性质。
In an earlier study, β3-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-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 β3-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 tRNACUA activated with each of four methyl-β-alanine isomers (1–4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated β-tyrosine moiety of β3-puromycin. Also conducted were a selection of clones that are responsive to β2-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-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.
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影响因子：
15
作者：
Lai, Yen-Ting;Tsai, Kuang-Lei;Sawaya, Michael R.;Asturias, Francisco J.;Yeates, Todd O.
通讯作者：
Yeates, Todd O.
影响因子：
2.1
作者：
BAIN, JD;WACKER, DA;CHAMBERLIN, AR
通讯作者：
CHAMBERLIN, AR
影响因子：
15
作者：
Hart, SA;Bahadoor, ABF;Schepartz, A
通讯作者：
Schepartz, A
影响因子：
14.8
作者：
Greenfield, Norma J.
通讯作者：
Greenfield, Norma J.
影响因子：
15
作者：
Cheng, RP;DeGrado, WF
通讯作者：
DeGrado, WF