Williams J M, Donly B C, Brown C M, Adamski F M, Trotman C N, Tate W P
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Eur J Biochem. 1989 Dec 22;186(3):515-21. doi: 10.1111/j.1432-1033.1989.tb15237.x.
A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. The nucleotide sequence preceding the in-phase stop codon within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that this pairing triggers the frameshift event. In vitro production of RNA coding for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence) region. The prokaryotic RF-2 gene and the gene for the other release factor, RF-1, which does not contain an in-frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA. These RF-1 and RF-2 RNAs both synthesized a major product of Mr approximately 45,000 when translated in vitro within reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules. The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site. The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system. Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of that of RF-1 RNA in the reticulocyte system. This was similar to the relative synthesis of the two proteins in a plasmid-DNA-directed prokaryotic transcription/translation system. These results show that in vitro on eukaryotic ribosomes where the Shine-Dalgarno-type interaction is not possible, high efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can still occur.
在大肠杆菌释放因子2（RF-2）的合成过程中，需要发生翻译移码以绕过编码序列中的一个框内终止密码子。RF-2 mRNA中同相位终止密码子之前的核苷酸序列与原核16S rRNA中发现的3' 反（Shine-Dalgarno序列）区域互补，并且Weiss等人（1988年）得出结论，这种配对触发了移码事件。体外产生编码RF-2的RNA，适合在真核核糖体上进行翻译，这使得能够测试真核核糖体是否能在此序列处发生移码。真核核糖体的18S rRNA不包含3' 反（Shine-Dalgarno序列）区域。将原核RF-2基因和另一种释放因子RF-1的基因（该基因不包含框内终止密码子）亚克隆到转录载体中，使得体外产生的RNA转录本类似于典型的真核mRNA。当在网织红细胞裂解物中进行体外翻译时，这些RF-1和RF-2 RNA都合成了一个主要产物，其分子量约为45,000；这是全长RF-1和RF-2分子预期的大小。RF-2产物被RF-2特异性抗体免疫沉淀，包括针对移码位点下游mRNA中编码的蛋白质区域的抗体。未检测到推测的过早终止产物，即一个25个氨基酸的寡肽，但一种化学合成的衍生物在翻译系统中显示出非常不稳定。因此，虽然无法计算移码的绝对效率，但在网织红细胞系统中，RF-2 RNA翻译的相对效率估计为RF-1 RNA的10-20%。这与质粒DNA指导的原核转录/翻译系统中两种蛋白质的相对合成情况相似。这些结果表明，在体外真核核糖体上，由于不可能发生Shine-Dalgarno型相互作用，RF-2 mRNA中同相位终止密码子周围仍可高效发生移码。
