Zhang S, Stancek M, Isaksson L A
Department of Microbiology, Stockholm University, S-106 91 Stockholm, Sweden.
Nucleic Acids Res. 1997 Nov 1;25(21):4301-6. doi: 10.1093/nar/25.21.4301.
A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.
已构建了一种在大肠杆菌中的顺式切割锤头状核酶（Rz）表达系统（3A'-Rz），可用于研究影响体内核酶切割的因素。核酶序列置于3A'mRNA的编码区，该mRNA由一个半合成翻译检测基因表达。测定了3'切割片段的大小和5'端序列，并通过定量引物延伸法测量了不同Rz变体的效率。结果表明，其中一种半活性构建体（3A'-RzIII）可作为核糖体的指示物，这些核糖体在mRNA中Rz锤头序列上游的终止密码子处通读或终止。未切割的mRNA中终止密码子的通读产生全长3A'蛋白。在核酶序列上游的终止密码子处终止产生缩短的终止产物。然而，由于自切割而应产生的mRNA片段并未产生任何可检测到的相应截短蛋白。除了对翻译核糖体的研究外，3A'-Rz系统还可用于分离因内部碱基改变或相互作用的宿主因子改变而导致核酶活性发生变化的突变菌株。
