Maizels N M
Proc Natl Acad Sci U S A. 1973 Dec;70(12):3585-9. doi: 10.1073/pnas.70.12.3585.
I have sequenced the first 63 bases of mRNA transcribed in vitro from the UV5 promoter mutant of the E. coli lactose operon. Sonic fragments of DNA, 1000 base pairs long and purified to contain only the lac operator-promoter region, were used as template. The UV5 promoter mutation allows transcription of the lac operon in the absence of catabolite activator protein and cAMP; lac repressor controls the synthesis of this RNA. I find that during synthesis, RNA polymerase pauses at particular sites along the DNA, naturally generating several discrete sizes of RNA that provide overlaps useful for sequencing. The UV5 lac mRNA initiates within the lac operator and copies the operator sequence. The AUG initiator codon for beta-galactosidase occurs at position 39 of the message. The sequence is: pppA-A-U-U-G-U-G-A-G-C-G-G-A-U-A-A-C-A-A-U-U-U- C-A-C-A-C-A-G-G-A-A-A-C-A-G-C-U-A-U-G-A-C-C-A-U- G-A-U-U-A-C-G-G-A-U-U-C-A-C-U-G-G.
我已经对从大肠杆菌乳糖操纵子的UV5启动子突变体体外转录的mRNA的前63个碱基进行了测序。使用了长度为1000个碱基对且经纯化仅包含乳糖操纵基因-启动子区域的DNA超声片段作为模板。UV5启动子突变使得乳糖操纵子在缺乏分解代谢物激活蛋白和cAMP的情况下进行转录;乳糖阻遏物控制这种RNA的合成。我发现,在合成过程中,RNA聚合酶会在沿着DNA的特定位点暂停,自然地产生几种不同大小的RNA,这些RNA提供了有助于测序的重叠区域。UV5乳糖mRNA在乳糖操纵基因内起始并复制操纵基因序列。β-半乳糖苷酶的AUG起始密码子出现在信使RNA的第39位。序列为:pppA-A-U-U-G-U-G-A-G-C-G-G-A-U-A-A-C-A-A-U-U-U-C-A-C-A-C-A-G-G-A-A-A-C-A-G-C-U-A-U-G-A-C-C-A-U-G-A-U-U-A-C-G-G-A-U-U-C-A-C-U-G-G。