BIOCHEMICAL AND MUTATIONAL ANALYSIS OF A PLANT-VIRUS POLYPROTEIN CLEAVAGE SITE
BIOCHEMICAL AND MUTATIONAL ANALYSIS OF A PLANT-VIRUS POLYPROTEIN CLEAVAGE SITE
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DOI：
10.1002/j.1460-2075.1988.tb02942.x
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发表时间：
1988-05-01
期刊：
影响因子：
11.4
通讯作者：
PARKS, TD
中科院分区：
文献类型：
--
作者：
DOUGHERTY, WG;CARRINGTON, JC;PARKS, TD
烟草蚀纹病毒（TEV）的RNA基因组被组织为编码346000（346 kd）mol. wt（Mr）多聚蛋白。346 kd的Mr多蛋白被49 kd的Mr病毒编码的蛋白酶在二肽Gln-Ser或Gln-Gly之间的五个不同位点切割。这些切割位点或基因产物边界由七肽序列定义。Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser或Gly.我们已经使用的54 kD先生核包涵体蛋白/30 kD先生衣壳蛋白连接作为一个模型来检查这些保守的氨基酸在定义一个切割位点的作用。推导了10个不同地理位置的TEV分离株的54 kd/30 kdMr蛋白裂解位点序列。保守的氨基酸存在于所有分离物中。为了确定这四种氨基酸是否是多蛋白底物活性的绝对要求，进行了定点突变分析。对编码TEV 54 kd/30 kd Mr基因产物切割位点的重组cDNA分子进行突变，并在无细胞系统中合成和加工多蛋白底物。在不同位置进行的单个氨基酸取代揭示了对天然保守氨基酸的强烈偏好。
The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus-encoded proteinase at five different sites between the dipeptides Gln-Ser or Gln-Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence...Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly.... We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site-directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell-free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.