Reddy R
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1988 Nov 5;263(31):15980-4.
U6 small nuclear RNA (snRNA), an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. In this study, the optimal conditions for transcription of the U6 snRNA gene in vitro were found to be similar to conditions optimal for transcription of 5S RNA genes. To purify the trans-acting factors necessary for the transcription of the U6 RNA gene, HeLa cell extracts were fractionated on a DEAE-Sephadex column, and three fractions, designated DE-50, DE-175, and DE-500, were obtained by stepwise elution with 50, 175, and 500 mM ammonium sulfate, respectively. DE-175 fraction transcribed tRNA and 5S RNA genes but not a mouse U6 RNA gene. Complementation of the DE-175 fraction with the DE-50 fraction resulted in the transcription of the U6 RNA gene. Experiments in which the transcription factor (TFIIIA) was selectively inactivated indicated that TFIIIA is not required for the transcription of the U6 RNA gene. These results show that the U6 snRNA gene, although transcribed by RNA polymerase III, differs from tRNA and 5S RNA genes in that factors other than TFIIIA, -IIIB, and -IIIC are required for U6 gene transcription in vitro.
U6小核RNA（snRNA）是真核生物剪接体的重要组成部分，其独特之处在于它由RNA聚合酶III合成，而所有其他U-snRNA均由RNA聚合酶II合成。U6基因以其功能性上游调控元件而闻名，这些元件类似于RNA聚合酶II调控序列基序。在本研究中，发现U6 snRNA基因体外转录的最佳条件与5S RNA基因转录的最佳条件相似。为了纯化U6 RNA基因转录所需的反式作用因子，将HeLa细胞提取物在DEAE-葡聚糖柱上进行分级分离，通过分别用50、175和500 mM硫酸铵逐步洗脱，获得了三个级分，分别命名为DE-50、DE-175和DE-500。DE-175级分可转录tRNA和5S RNA基因，但不能转录小鼠U6 RNA基因。DE-175级分与DE-50级分互补可导致U6 RNA基因的转录。使转录因子（TFIIIA）选择性失活的实验表明，U6 RNA基因的转录不需要TFIIIA。这些结果表明，U6 snRNA基因虽然由RNA聚合酶III转录，但与tRNA和5S RNA基因不同，体外U6基因转录需要TFIIIA、-IIIB和-IIIC以外的因子。
