Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of EScherichia coli.
Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of EScherichia coli.
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DNA 碱基类似物对大肠杆菌色氨酸操纵子衰减子转录终止的影响。
DOI：
10.1073/pnas.79.4.998
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发表时间：
1982
影响因子：
11.1
通讯作者：
Platt,T
中科院分区：
文献类型：
--
作者：
Farnham,PJ;Platt,T
我们设计了一种方法，通过对携带克隆的trp衰减子DNA的噬菌体M13衍生物进行引物合成，在体外将脱氧核糖核苷酸碱基类似物特异性掺入色氨酸（trp）操纵子衰减子区域的任一链中。我们利用这些技术来扩展先前涉及转录终止中 RNA-RNA 和 RNA-DNA 相互作用的研究，试图确定终止区域中模板 DNA 分子的贡献性质。一般来说，我们发现当相似的类似物掺入 DNA 时，不会发生碱基类似物掺入 mRNA 时所观察到的转录终止的显着影响。只有增强 A·T 或 A·U 碱基配对的类似物 2,6-二氨基嘌呤脱氧核糖核苷酸三磷酸 (dDapTP) 才会引发显着反应：在模板 DNA 链中，该类似物的存在会增加 thetrpattenuator 的通读。类似物 5-溴尿嘧啶脱氧核糖核苷三磷酸 (BrdUTP) 也可增强与其互补碱基的配对，当将其置于 thetrpattenuator 或突变体 attenuatortrp a1419 的任一链中时，对终止没有可检测到的影响。令人惊讶的是，虽然类似物 5-碘胞嘧啶脱氧核糖核苷三磷酸 (IdCTP) 不影响终止，但它对转录起始、抑制 trp 启动子活性以及刺激其他区域的转录有很大影响。这些结果支持假设的 mRNA 末端尿苷与模板 DNA 链之间在增强终止方面的相互作用，并表明 DNA 没有显着的额外贡献。此外，M13衍生物在制备规模上将类似物掺入DNA的新用途提供了一种以一般但受控的方式引入突变的技术，作为研究其他调控区域的新手段。
We have devised a method to specifically incorporate deoxyribonucleotide base analogsin vitrointo either strand of the tryptophan (trp) operon attenuator region, using primed synthesis on bacteriophage M13 derivatives carrying clonedtrpattenuator DNA. We have employed these techniques to extend previous studies implicating both RNA-RNA and RNA-DNA interactions in transcription termination in an attempt to determine the nature of the contribution from the template DNA molecule in termination regions. In general, we find that the dramatic effects upon transcription termination seen with base analog incorporation into mRNA do not occur when similar analogs are incorporated into the DNA. Only the analog 2,6-diaminopurine deoxyribonucleotide triphosphate (dDapTP), which strengthens A·T or A·U base pairing, elicits a significant response: in the template DNA strand, the presence of this analog increases read-through at thetrpattenuator. The analog 5-bromouracil deoxyribonucleoside triphosphate (BrdUTP), which also strengthens pairing with its complementary base, has no detectable effect on termination when it is placed in either strand of thetrpattenuator or the mutant attenuatortrp a1419. Surprisingly, though the analog 5-iodocytosine deoxyribonucleoside triphosphate (IdCTP) does not affect termination, it has a great effect on initiation of transcription, depressingtrppromoter activity as well as stimulating transcription from other regions. These results support the postulated interaction between terminal uridines in mRNA and the template DNA strand in enhancing termination and suggest that there are no significant additional contributions from the DNA. In addition, the novel use of M13 derivatives for incorporating analogs into the DNA on a preparative scale provides a technique for introducing mutations in a general but controlled fashion as a new means for studying other regulatory regions.