McConkey G A, Bogenhagen D F
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794.
Genes Dev. 1988 Feb;2(2):205-14. doi: 10.1101/gad.2.2.205.
Current models for the differential control of expression of Xenopus somatic and oocyte 5S RNA genes suggest that an impaired ability to bind TFIIIA contributes to the inactivation of oocyte 5S RNA genes in somatic cells. The somatic 5S RNA gene is transcribed more efficiently than the major oocyte 5S RNA gene in S-150 extracts of mature oocytes. However, this differential transcription efficiency is not determined simply by the relative affinity for binding of a positive transcription factor, TFIIIA. We have compared the abilities of somatic, major oocyte, and minor oocyte 5S RNA genes to bind TFIIIA using both a standard footprint competition assay and an indirect DNase protection assay. This indirect DNase protection assay permits the direct comparison of TFIIIA binding to two templates in one reaction. Both assay methods indicate that the major oocyte 5S RNA gene and the somatic 5S RNA gene bind TFIIIA with equal affinity. As a further control, we have confirmed earlier work indicating that the minor oocyte gene binds TFIIIA with a reduced affinity. Binding of TFIIIA to these three 5S RNA genes results in a different pattern of protection of each gene. We suggest that slight differences in the contacts between TFIIIA and the 5' border of the control region influence the ability of additional transcription factors to bind to the TFIIIA:5S DNA complex.
非洲爪蟾体细胞和卵母细胞5S RNA基因表达差异调控的现有模型表明，结合TFIIIA能力受损是导致卵母细胞5S RNA基因在体细胞中失活的原因。在成熟卵母细胞的S-150提取物中，体细胞5S RNA基因的转录效率高于主要的卵母细胞5S RNA基因。然而，这种转录效率差异并非简单地由正转录因子TFIIIA的相对结合亲和力决定。我们使用标准的足迹竞争试验和间接DNase保护试验，比较了体细胞、主要卵母细胞和次要卵母细胞5S RNA基因结合TFIIIA的能力。这种间接DNase保护试验允许在一个反应中直接比较TFIIIA与两个模板的结合情况。两种试验方法均表明，主要卵母细胞5S RNA基因和体细胞5S RNA基因与TFIIIA的结合亲和力相同。作为进一步的对照，我们证实了早期的研究结果，即次要卵母细胞基因与TFIIIA的结合亲和力较低。TFIIIA与这三种5S RNA基因的结合导致了每个基因不同的保护模式。我们认为，TFIIIA与控制区5'边界之间接触上的细微差异会影响其他转录因子与TFIIIA:5S DNA复合物结合的能力。
