CONTRIBUTIONS OF DISCRETE TRANSFER RNA(SER) DOMAINS TO AMINO-ACYLATION BY ESCHERICHIA-COLI SERYL-TRANSFER RNA-SYNTHETASE - A KINETIC-ANALYSIS USING MODEL RNA SUBSTRATES
CONTRIBUTIONS OF DISCRETE TRANSFER RNA(SER) DOMAINS TO AMINO-ACYLATION BY ESCHERICHIA-COLI SERYL-TRANSFER RNA-SYNTHETASE - A KINETIC-ANALYSIS USING MODEL RNA SUBSTRATES
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DOI：
10.1093/nar/21.19.4467
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发表时间：
1993-09-25
影响因子：
14.9
通讯作者：
SAKS, ME
中科院分区：
文献类型：
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作者：
SAMPSON, JR;SAKS, ME
使用纯化的大肠杆菌测定了代表大肠杆菌丝氨酸tRNA确定区域的T7转录物的氨酰化动力学。大肠杆菌丝氨酸-tRNA合成酶（SerRS）的动力学值被用来估计各种tRNA（ser）结构域对SerRS识别的相对贡献。分析表明，额外的茎/环结构，特征的II型tRNA，如tRNA（Ser），是域，使最大的贡献K（猫）/K（m）的氨酰化。当额外的茎/环变成I型tRNA额外环的共有序列时，氨酰化的K（m）增加了约1 000倍，表明茎结构对tRNA（Ser）与SerRS的结合有重要作用。仅代表tRNA（ser）同轴受体-TpsiC茎/环结构域的模型RNA也被SerRS特异性氨酰化，其k（cat）/K（m）比背景水平高约1000倍。该结构域对氨酰化的贡献的很大一部分归因于受体茎序列，使受体茎成为SerRS识别的第二重要结构域。最后，当tRNA（ser）的整个反密码子茎/环被删除时，k（cat）/K（m）基本上不变，表明反密码子核苷酸和周围的茎/环结构对于SerRS的识别都不重要。
The aminoacylation kinetics of T7 transcripts representing defined regions of Escherichia coli serine tRNAs were determined using purified E. coli seryl-tRNA synthetase (SerRS) and the kinetic values were used to estimate the relative contribution of various tRNA(ser) domains to recognition by SerRS. The analysis revealed that the extra stem/loop structure, characteristic of type II tRNAs such as tRNA(Ser), is the domain which makes the largest contribution to K(cat)/K(m) of aminoacylation. Moreover, K(m) of aminoacylation was increased by a factor of about 1 000 when the extra stem/loop was changed to the consensus sequence of type I tRNA extra loops indicating that the stem structure contributes significantly to the binding of tRNA(Ser) to SerRS. A model RNA, which represents only the tRNA(ser) coaxial acceptor-TpsiC stem/loop domain, was also specifically aminoacylated by SerRS having a k(cat)/K(m) about 1000-fold greater than background levels. A significant portion of the contribution of this domain to aminoacylation is attributable to the acceptor stem sequence making the acceptor stem the second most important domain for recognition by SerRS. Finally, k(cat)/K(m) was essentially unchanged when the entire anticodon stem/loop of tRNA(ser) was deleted indicating that neither the anticodon nucleotides nor the surrounding stem/loop structure are important for recognition by SerRS.