Spanjaard R A, van Dijk M C, Turion A J, van Duin J
Department of Biochemistry, University of Leiden, The Netherlands.
Gene. 1989 Aug 15;80(2):345-51. doi: 10.1016/0378-1119(89)90298-9.
A synthetic ribosome-binding site (RBS) containing a 7-nucleotide-long Shine-Dalgarno (SD) sequence was placed ahead of the rat interferon (IFN)-alpha 1 coding region. The translational efficiency of this construct was extremely low. Structural probing of transcripts with RNases T1 and U2 combined with computer predictions revealed the presence of a stable hairpin in which the SD region was base-paired to codons 3, 4 and 5 of the IFN mRNA. Each mutation in this stem changing an A-U to an A.C or a G-C a G.U pair increased translational efficiency about fourfold and this effect could be reversed by a compensating stabilizing substitution in the other strand of the stem. We conclude that the strength of an RBS is to a major degree determined by its involvement in secondary structure. We also show that the negative effect of secondary structure on the efficiency of an RBS can be overcome by allowing upstream translation to terminate within the base-paired region. In our clones, termination-dependent restarts occur at a frequency comparable to that taking place in constructs containing destabilized hairpins.
一个含有7个核苷酸长的Shine-Dalgarno（SD）序列的合成核糖体结合位点（RBS）被置于大鼠干扰素（IFN）-α1编码区之前。该构建体的翻译效率极低。用核糖核酸酶T1和U2对转录本进行结构探测并结合计算机预测，发现存在一个稳定的发夹结构，其中SD区域与IFN mRNA的第3、4和5密码子碱基配对。该茎中的每个将A-U变为A.C或G-C、G-U对的突变都会使翻译效率提高约四倍，并且这种效应可以通过茎的另一条链中的补偿性稳定取代来逆转。我们得出结论，RBS的强度在很大程度上取决于其参与二级结构的程度。我们还表明，通过允许上游翻译在碱基配对区域内终止，可以克服二级结构对RBS效率的负面影响。在我们的克隆中，依赖终止的重新起始发生的频率与在含有不稳定发夹结构的构建体中发生的频率相当。
