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作 者:顾利群[1] 刘士辉[1] 熊凌霜[1] 黄培堂[1]
机构地区:[1]军事医学科学院生物工程研究所
出 处:《生物化学杂志》1994年第1期64-69,共6页
摘 要:序列分析中发现tPAK2编码区结构基因中存在一段反转重复序列,富含G、C。利用计算机预测tPAmRNA二级结构证明tPAmRNA在此处可以形成△Gm=-25.5KCal/mol的发夹结构。本研究利用定点突变技术消除了这段反转重复序列,在大肠杆菌中进行了消除前后tPA转录和翻译水平的比较。结果表明消除之后,细菌总RNA中tPA特异mRNA含量减少,细菌表达产物中tPA蛋白占破菌沉淀物的百分比却基本不变。提示大肠杆菌中基因编码区mRNA二级结构一般不构成转录的终止,但有利于mRNA的稳定性,对翻译表达无影响。DNA sequencing showed that an inverted repeat sequence existed in tPA K2 coding region.It was also identified by computer as the only moderately stable hairpin(△Gm= -25. 5KCal/mol)in tPA mRNA. In these experiments, the inverted repeat sequence was eliminated by sitedirected mutagenesis. And by the methods of RNA dot blot hybridization and SDS-PAGE ,the influence of the inverted repeat sequence on tPA transcription and translation was studied in E. Coli. The results showed that after the elimination,the relative amount of tPA mRNA in total RNA decreased two folds and the yield of tPA product in bacteria extract maintained the same.This revealed that secondary structure of mRNA in protein coding region generally didn't terminate transcription in prokaryote,and had no effect on translational efficiency.But it was of benefit to the stability of mRNA.
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