Roman J, Rubin M N, Woodson S A
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742-2021, USA.
RNA. 1999 Jan;5(1):1-13. doi: 10.1017/s1355838299981244.
Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.
I组内含子的反向剪接被认为是一种将内含子序列转移到新基因的机制。通过反向剪接将嗜热四膜虫内含子整合到大肠杆菌23S rRNA中，这取决于内含子的引导序列与靶位点之间的碱基配对。为了研究反向剪接的底物特异性，在大肠杆菌中表达了具有不同引导序列的野生型和18个突变内含子。通过RT-PCR扩增内含子-rRNA连接点，发现在23S rRNA的69个位点有部分反向剪接，在一个新位点有完全整合。在一些潜在的靶位点未观察到反向剪接，而23S rRNA的其他区域比预期更具反应性。结果表明，反向剪接的频率受rRNA结构的调节。与嗜热四膜虫26S rRNA的天然剪接连接点同源的位点相比，23S rRNA结构域V中的一个新整合位点（U2074）在大肠杆菌中进行内含子剪接的效率低10倍，这表明该位点不太有利于正向反应。
