POLY(A) RNA IN ESCHERICHIA-COLI - NUCLEOTIDE-SEQUENCE AT THE JUNCTION OF THE LPP TRANSCRIPT AND THE POLYADENYLATE MOIETY
POLY(A) RNA IN ESCHERICHIA-COLI - NUCLEOTIDE-SEQUENCE AT THE JUNCTION OF THE LPP TRANSCRIPT AND THE POLYADENYLATE MOIETY
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DOI：
10.1073/pnas.89.16.7546
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发表时间：
1992-08-15
影响因子：
11.1
通讯作者：
SARKAR, N
中科院分区：
文献类型：
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作者：
CAO, GJ;SARKAR, N
虽然人们已经知道细菌mRNA分子在其3'端携带聚腺苷酸基团，但对细菌poly(A) RNA的分子结构一无所知。为了确定特定细菌mRNA的聚腺苷化位点，我们利用缺乏外核糖核酸酶的大肠杆菌细胞中poly(a) RNA水平升高的优势，利用禽成髓细胞病病毒逆转录酶和含有寡核苷酸（dT）的引物合成了由lpp基因编码的多腺苷化脂蛋白mRNA的DNA互补。利用聚合酶链反应和合适的寡核苷酸引物扩增cDNA的5′端片段，将扩增的DNA克隆于pUC18中，进行核苷酸序列分析。发现有4个克隆包含lpp mRNA的整个3'端编码区，poly(A)附着在转录物下游非翻译区两个位点中的任意一个上。在一种类型的克隆中，多腺苷酸片段附着在lpp mRNA的推定转录终止位点，而其他克隆缺乏rho独立转录终止端的茎环结构，多腺苷酸片段附着在初级转录物末端茎环的残基上。提出了细菌mRNA的聚腺苷化模型，其中聚(A)聚合酶和外切酶竞争mRNA分子的3'端。
Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA. To define the polyadenylylation site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coli deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-containing primer. The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis. Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript. In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the rho-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-loop of the primary transcript. A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonucleases compete for the 3' ends of mRNA molecules.