在酵母体内用RNA聚合酶Ⅱ表达大肠杆菌tRNA^（Sec）基因  

Transcription of E.coli tRNA￣(Sec) Gene by RNA Polymerase Ⅱ in S.cerevisiae

作　　者：张妙华[1] 刘建华[1] 金由辛[1] 

机构地区：[1]中国科学院上海生物化学研究所分子生物学国家重点实验室

出　　处：《生物化学与生物物理学报》1995年第5期529-536,共8页

基　　金：国家"863"高科技基金;国家自然科学基金

摘　　要：我们将大肠杆菌的硒代半胱氨酸ｔＲＮＡ基因（ＳｅｌＣ基因）连接到分泌型表达质粒ＰＶＴ１０２Ｕ－αＭＦＬ中，并调整好阅读框架，使蛋白翻译的终止密码子位于ＳｅｌＣ基因的下游．将该质粒转化酵母，通过ＳＤＳ－ＰＡＧＥ分析，在ＳＤ液体培养基中检测出７～８ｋｄ的蛋白条带，这与理论值是相符合的。同时，我们抽提出酵母的总ＲＮＡ用互补与该ｔＲＮＡＴＣ茎环区的２１－ｍｅｒ寡核苷酸，经５′标记后作为探讨进行Ｎｏｒｔｈｅｒｎｂｌｏｔ。结果有两条较强的条带和一条较弱的条带，较强条带中一条相当于７９０ｎｔｓ左右，另一条相当于３７０ｎｔｓ左古，根据组建的表达型质粒结构资料推测７９０ｎｔｓ左右的条带是由ＲＮＡ聚合酶ＩＩ转录的未被加工的前体；３７０ｎｔｓ左右的条带是５′端被加工而３′端未被加工的分子，较弱的条带则是相当于９０ｎｔｓ左右的成熟ｔＲＮＡ分子。因此，我们认为ＲＮＡ聚合酶ＩＩ可以转录ｔＲＮＡ基因。由于实验用酵母的３′内切核酸酶含量较低，致使带长尾巴的ｔＲＮＡ前体３′端加工速度缓慢。The selenocysteine tRNA(SelC)gene of E.coli was inserted into a sereefory expression plasmid vector,PVT102U-αMFL,with adjusted reading frame of protein translation,and added stop codon at the downstream of the structure gene.Its expression was established in Saccharomyce cerevisiae.A 7-8kd band in SD culture media was shown by SDS-PAGE analysis to be consistent with the theoretical value.The total RNA was extracted from yeast and Northern blot was done with a 5'end-labeled 21-mer oligonucleotide probe complementary to the TC region of the tRNA￣(sec).The result showed that beside one weak band there were two strong bands,one of which corresponded fo 790mts and the other,370nts.It is suggested that the 790nts band is the unprocessed precursor.of RNA polymerase II-mediated transcript,the 370nts band is RNA molecules processed at the 5'-ends but not the 3'-ends,and the weaker band is the mature tRNA of about 90nts long.We concluded that RNA polII could transcribe tRNA genes.Because of the low level of 3'-endonuclease in the yeast strain we used,the 3'-end processing of the precursors with long tails was slow.

关 键 词：酵母菌 RNA聚合酶II tRNA^Sec基因 大肠杆菌 

分 类 号：Q939.506[生物学—微生物学]