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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.
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Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

机译：小RNA发夹在噬菌体λ的转录抗终止中的双重功能。

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Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.
机译：噬菌体λ早期操纵子中下游基因的转录需要一个称为螺母的启动子近端元件。该位点以RNA的形式顺式作用以组装转录抗终止复合物，该复合物由λN蛋白和至少四个宿主因子组成。坚果位点RNA包含一个称为boxB的小茎环结构。在这里，我们显示boxB RNA以高亲和力和特异性结合N蛋白。虽然N的结合仅限于茎环的5'子域，但特定的N识别依赖于完整的茎环结构和五聚体环中的两个关键核苷酸。这些核苷酸的取代影响N结合和抗终止。值得注意的是，其他环核苷酸的取代在体内也减少了抗终止作用，但它们在体外对N的结合没有可检测的作用。这些3'环突变体无法在具有RNA聚合酶（RNAP），N和宿主因子NusA的最小系统中支持抗终止作用。此外，这些突变体减弱了NusA刺激RNAP-boxB-N复合物形成的能力。因此，我们建议boxB RNA在抗终止中执行两个关键功能。首先，boxB与N结合并固定在RNAP附近以增强它们之间的相互作用，大概是通过增加N的局部浓度来实现的。其次，boxB与NusA合作，最有可能使N和RNAP紧密接触并将RNAP转化为耐末端的州。

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