Andrews D L, Millstein L, Hamkalo B A, Gottesfeld J M
Nucleic Acids Res. 1984 Oct 25;12(20):7753-69. doi: 10.1093/nar/12.20.7753.
We have constructed hybrid plasmids bearing both Xenopus 5S RNA genes and satellite I sequences in order to test the effect of satellite DNA on 5S gene transcription. Satellite sequences inactivate 5S transcription in both HeLa S100 and Xenopus oocyte microinjection transcription assays. Inactivation of 5S transcription by satellite DNA is observed both in cis and in trans. Transcription of a tRNA gene is also precluded by satellite I DNA. The Xenopus satellite I repeat contains an RNA polymerase III transcription unit which is highly active in both assay systems. This promoter element is 10- to 25-fold more efficient than the 5S gene in transcription competition assays. This quantitative difference in affinity for transcription components may explain the inactivation of 5S transcription by satellite sequences. The satellite I promoter forms stable transcription complexes in vitro which do not dissociate for at least 30 rounds of transcription. Although stable complex formation on the satellite promoter is largely temperature independent over the range of 0-20 degrees, complex formation on both 5S and tRNA genes exhibits a steep temperature dependence characteristic of DNA helix unwinding. The DNA sequence requirements for stable complex formation on 5S genes have been determined using 5' deletion mutants.
为了测试卫星DNA对5S基因转录的影响，我们构建了同时携带非洲爪蟾5S RNA基因和卫星I序列的杂种质粒。在HeLa S100细胞和非洲爪蟾卵母细胞显微注射转录试验中，卫星序列均使5S转录失活。卫星DNA对5S转录的失活作用在顺式和反式中均能观察到。卫星I DNA也会阻止tRNA基因的转录。非洲爪蟾卫星I重复序列包含一个RNA聚合酶III转录单位，该单位在两种试验系统中均具有高活性。在转录竞争试验中，该启动子元件的效率比5S基因高10至25倍。对转录成分亲和力的这种数量差异可能解释了卫星序列对5S转录的失活作用。卫星I启动子在体外形成稳定的转录复合物，至少经过30轮转录才会解离。虽然卫星启动子上稳定复合物的形成在0至20度的范围内很大程度上不依赖温度，但5S和tRNA基因上复合物的形成表现出DNA螺旋解旋所特有的强烈温度依赖性。利用5'缺失突变体已确定了5S基因上形成稳定复合物的DNA序列要求。
