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首页> 外文期刊>Molecular and Cellular Biology >Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.
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Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.

机译：鉴定与单纯疱疹病毒1型胸苷激酶前体RNA的加工和聚腺苷酸化相关的复合物。

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摘要

Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.
机译：在HeLa细胞核提取物中检查了含有1型单纯疱疹病毒（HSV-1）胸苷激酶（tk）基因聚腺苷酸化信号区域的底物RNA的裂解和聚腺苷酸化。 3'-端RNA处理准确高效，需要ATP和Mg2 +。在EDTA存在下，或者当ATP被3'dATP（cordycepin）或AMP（CH2）PP（ATP的不可水解类似物）替代时，发生了裂解，但未发生聚腺苷酸化。体外和体内加工显示出相同的信号元素要求：一系列包含接头扫描，内部缺失和小的插入突变的底物以相同的相对效率在体外和体内的相同位置进行加工。通过凝胶迁移率移动分析鉴定了参与3'-端RNA加工的复合物。该复合物迅速形成，在20至30分钟后达到最高水平，并在2小时后大量还原。在0℃下或在缺少聚腺苷酸化信号的底物下形成的复合物非常少。通过添加过量的35S标记的tk或腺病毒L3前体RNA，可以防止32P标记的tk底物进入复合物。缺乏完整的聚腺苷酸化信号的tk RNA未观察到竞争。

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