Sastry S S, Hoffman P L
Rockefeller University, New York, New York 10021, USA.
Biochem Biophys Res Commun. 1995 Jun 6;211(1):106-14. doi: 10.1006/bbrc.1995.1784.
It was previously thought that elongating Escherichia coli transcription ternary complex consists of an RNA polymerase molecule enclosing 17 +/- 1 melted bases (bubble) of the template DNA and a 12-base-pair RNA-DNA hybrid ("transcription bubble paradigm"). Recent evidence suggests that ternary elongation complexes are heterogeneous and possibly vary in bubble size and length of RNA-DNA hybrid. We used a new type of assay to address the relative contributions of bubble size, secondary structure of RNA and RNA-DNA hybrid length during elongation. Synthetic RNA-DNA bubble duplexes are assembled in vitro. RNA structure 5' to the RNA-DNA hybrid, hybrid length and bubble size are systematically changed. The relative efficiency of E. coli and T7 RNA polymerases to elongate RNA primer is quantitated. RNA elongation was high (approximately 22-30%) when a stable hairpin was present towards the 5' end of the primer. Efficiency of elongation was lower for RNA primers without hairpins. Hairpin RNAs with presumed RNA-DNA hybrids of 3-7 bp were efficiently elongated compared to hairpins that presumably form 10bp hybrids. Preformed bubbles of different sizes (2,5 or 20 bases) were functional in all cases where elongation was moderate or high. We concluded that RNA secondary structure plays a dominant role compared to hybrid length or bubble size in determining efficient elongation by RNA polymerases.
以前人们认为，延伸中的大肠杆菌转录三元复合物由一个RNA聚合酶分子组成，该分子包围着模板DNA的17±1个解链碱基（泡）和一个12碱基对的RNA-DNA杂交体（“转录泡范式”）。最近的证据表明，三元延伸复合物是异质的，并且泡大小和RNA-DNA杂交体长度可能有所不同。我们使用了一种新型检测方法来研究延伸过程中泡大小、RNA二级结构和RNA-DNA杂交体长度的相对贡献。合成的RNA-DNA泡双链体在体外组装。RNA-DNA杂交体5'端的RNA结构、杂交体长度和泡大小被系统地改变。对大肠杆菌和T7 RNA聚合酶延伸RNA引物的相对效率进行定量。当引物的5'端存在稳定的发夹结构时，RNA延伸率很高（约22-30%）。没有发夹结构的RNA引物的延伸效率较低。与可能形成10bp杂交体的发夹相比，具有3-7bp假定RNA-DNA杂交体的发夹RNA能被有效延伸。在延伸适度或较高的所有情况下，不同大小（2、5或20个碱基）的预制泡都具有功能。我们得出结论，在决定RNA聚合酶的有效延伸方面，RNA二级结构比杂交体长度或泡大小起主导作用。
