He B, Rong M, Durbin R K, McAllister W T
Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY Health Science Center at Brooklyn, NY 11203, USA.
J Mol Biol. 1997 Jan 24;265(3):275-88. doi: 10.1006/jmbi.1996.0741.
We have identified a mutation (E148A) in T7 RNA polymerase (RNAP) that results in an enzyme which aborts transcription primarily when the nascent RNA achieves a length of 5 nt. This phenomenon is observed at a consensus promoter, but is even more strongly observed at promoters that are altered in the initiation region. Although the abortive product is of a fixed length (5 nt), the positions of the base substitutions in the initiation region that enhance this effect do not appear to be fixed, and we have observed the effect with a variety of initiation-region promoter variants. The phenomenon is also observed during promoter-independent transcription when transcribing a homopolymeric template such as poly(dC). Under conditions where the active site of the RNAP cannot extend beyond the third nucleotide in the template strand and the maximum length of the RNA:DNA hybrid cannot exceed three base-pairs (i.e. when synthesizing oligoG products due to transcript slippage at a promoter that initiates with the sequence +1 GGG...) the mutant RNAP gives rise to a normal spectrum of products 2 to 14 nt in length with no evidence of a block at 5 nt. Neither promoter binding nor promoter melting appears to be involved in this phenotype, as the mutant RNAP binds normally to promoter sequences and the behavior of the enzyme is unaffected by removal of the non-template strand in the initiation region of the promoter or on a supercoiled template. Importantly, the mutant RNAP is defective in binding single strand oligomers of RNA. These results suggest that the affected region of the RNAP may form part of the RNA product binding site and may be involved in the transition from an unstable initiation complex to a stable elongation complex, perhaps by sensing the presence of a nascent RNA and/or RNA:DNA hybrid.
我们在T7 RNA聚合酶（RNAP）中鉴定出一种突变（E148A），该突变产生的一种酶主要在新生RNA达到5个核苷酸长度时中止转录。这种现象在共有启动子处可以观察到，但在起始区域发生改变的启动子处观察到的更为明显。尽管流产产物的长度是固定的（5个核苷酸），但起始区域中增强这种效应的碱基替换位置似乎并不固定，并且我们在多种起始区域启动子变体中都观察到了这种效应。在转录同聚物模板（如聚（dC））时，在不依赖启动子的转录过程中也观察到了这种现象。在RNAP的活性位点无法延伸到模板链上第三个核苷酸之外且RNA:DNA杂交体的最大长度不能超过三个碱基对的条件下（即由于在以序列+1 GGG...起始的启动子处转录滑移而合成oligoG产物时），突变型RNAP产生长度为2至14个核苷酸的正常产物谱，没有5个核苷酸处受阻的迹象。这种表型似乎与启动子结合或启动子解链无关，因为突变型RNAP能正常结合启动子序列，并且去除启动子起始区域或超螺旋模板上的非模板链不会影响该酶的行为。重要的是，突变型RNAP在结合RNA单链寡聚物方面存在缺陷。这些结果表明，RNAP受影响的区域可能构成RNA产物结合位点的一部分，并且可能参与从不稳定的起始复合物到稳定的延伸复合物的转变，也许是通过感知新生RNA和/或RNA:DNA杂交体的存在来实现的。
