Skolnik-David H, Aloni Y
Department of Genetics, Weizmann Institute of Science, Rehovot, Israel.
EMBO J. 1983;2(2):179-84. doi: 10.1002/j.1460-2075.1983.tb01402.x.
Viral transcription complexes were isolated from SV40-infected cells and incubated in vitro in the presence of [alpha-32P]UTP to allow elongation of the promoter-proximal RNA up to the attenuation sites. The 94 nucleotide attenuated RNA (spanning nucleotides 243-336) was purified, digested with RNase T1 and fingerprinted. The labeled oligonucleotides were then isolated, digested with RNase T2 and their base composition was determined. Based on these analyses 10 consecutive oligonucleotides, spanning residues 259-336, were identified. As the in vivo synthesized oligonucleotides are unlabeled the junctions between labeled and unlabeled oligonucleotides define the in vivo pause sites of RNA polymerase molecules. The characterization of the 10 radioactive spots and their relative intensities allowed the localization of two in vivo pause sites: one at 13-16 nucleotides downstream from the major initiation site presumably at the initial opening of the DNA helix and the second at approximately 40 nucleotides downstream from the major initiation site, just past a GC-rich region of dyad symmetry. It is postulated that pausing of RNA polymerase molecules in the leader region is an essential process in the control of SV40 late transcription.
从感染SV40的细胞中分离出病毒转录复合物，并在[α-32P]UTP存在的情况下进行体外孵育，以使启动子近端RNA延伸至衰减位点。纯化94个核苷酸的衰减RNA（跨越核苷酸243 - 336），用核糖核酸酶T1消化并进行指纹分析。然后分离标记的寡核苷酸，用核糖核酸酶T2消化并确定其碱基组成。基于这些分析，鉴定出了跨越残基259 - 336的10个连续寡核苷酸。由于体内合成的寡核苷酸未标记，标记和未标记寡核苷酸之间的连接点定义了RNA聚合酶分子在体内的暂停位点。对10个放射性斑点及其相对强度的表征确定了两个体内暂停位点：一个在主要起始位点下游13 - 16个核苷酸处，大概在DNA螺旋的初始打开处；另一个在主要起始位点下游约40个核苷酸处，刚好在一个富含GC的二元对称区域之后。据推测，RNA聚合酶分子在前导区的暂停是SV40晚期转录调控中的一个重要过程。
