RNASE III-DEPENDENT HYDROLYSIS OF LAMBDA-CII-O GENE MESSENGER-RNA MEDIATED BY LAMBDA-OOP ANTISENSE RNA
RNASE III-DEPENDENT HYDROLYSIS OF LAMBDA-CII-O GENE MESSENGER-RNA MEDIATED BY LAMBDA-OOP ANTISENSE RNA
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DOI：
10.1101/gad.4.12a.2223
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发表时间：
1990-12-01
影响因子：
10.5
通讯作者：
WULFF, DL
中科院分区：
文献类型：
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作者：
KRINKE, L;WULFF, DL
噬菌体的77个核苷酸OOP反义RNA。补充.lambda。cII-O mRNA位于cII基因3'端包含55个核苷酸，cII和O基因之间的顺反子间区域包含22个核苷酸的区域。由多拷贝质粒产生的OOP RNA可抑制.lambda。.apprx表达cII基因。100倍通过RNase iii依赖机制。利用引物延伸分析从诱导的.lambda中分离的细胞RNA。我们在含有OOP DNA质粒的cII-O mRNA中发现了一个与OOP RNA互补区域的切割位点，距离该区域的teh3”端有13个核苷酸。核糖核酸酶保护实验表明，当OOP RNA在RNase III+细胞中过量产生时，该重叠区域的几乎所有cII-O mRNA都被切割，而在RNase III-细胞中则没有。可以检测到从切割位点延伸到O基因的RNA片段，而延伸到cII基因的姊妹片段则无法检测到，必须通过额外的水解事件消除，RNase II+和RNase III-细胞之间的未切割mRNA水平差异要小得多，在目标区域两侧的数百个核苷酸处。另一种依赖于ooprna的水解过程发生在RNase III-细胞中，导致两个区域中的一个发生裂解，一个在RNase III+细胞中观察到的裂解位点附近，第二个在OOPRNA和cII-O mRNA之间互补区域末端以外的几个核苷酸。在后一种情况下，延伸到cII基因的片段是稳定的，而姊妹O基因片段被破坏，与RNase iii依赖的过程直接相反。
The 77-nucleotide OOP antisense RNA of bacteriophage .lambda. complements .lambda.cII-O mRNA in a region that includes 55 nucleotides at the 3'' end of the cII gene and 22 nucleotides in the intercistronic region between the cII and O genes. OOP RNA, produced from multicopy plasmids, inhibits .lambda. cII gene expression by .apprx. 100-fold through an RNase III-dependent mechanism. Using primer extension analysis of cellular RNA isolated from an induced .lambda. lysogen that contains an OOP DNA plasmid, we have identified a cleavage site in cII-O mRNA within the regin of complementarity with OOP RNA, at 13 nucleotides from teh3'' end of that region. Ribonuclease protection experiments demonstrate that almost all cII-O mRNA in this overlap region is cleaved when OOP RNA is overproduced in RNase III+ cells but not in RNase III- cells. RNA fragments are detected that extend into the O gene from the cleavage sites, while the sister fragments that extend into the cII gene cannot be detected and must be eliminated by additional hydroytic events Differences in leels of uncleaved mRNA between RNase II+ and RNase III- cells are much less at several hundred nucleotides to either side of the target region. An alternate OOPRNA-dependnet hydrolytic process occurs in RNase III- cells that results in cleavages in one of two regions, one close to the cleavage site observed in RNase III+ cells, and the second several nucleotides beyond the end of the complementary region between OOP RNA and cII-O mRNA. In this latter case, the fragments that extend into the cII gene are stable, while the sister O gene fragments are destroyed, in direct contrast to the RNase III-dependent process.