Gao W, Tyagi S, Kramer F R, Goldman E
Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark 07103, USA.
Mol Microbiol. 1997 Aug;25(4):707-16. doi: 10.1046/j.1365-2958.1997.5081871.x.
In '5'-translational blockage', significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons near the translation start prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes and monosomes and a ribosome-free pool. New hybridization probes, called 'molecular beacons', and standard slot blots were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present in the message compared with when high-usage codons were present. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted or when the arginine codons were inserted near the 3' end. These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.
在“5'端翻译阻断”过程中，当在信使RNA的翻译起始位点附近插入连续的低使用频率密码子时，大肠杆菌中蛋白质的合成产量会显著降低（而当这些相同密码子插入下游时，产量降低或无影响）。我们检验了这样一种假说，即核糖体在翻译起始位点附近遇到这些低使用频率密码子时会过早释放信使RNA。将来自多核糖体梯度的RNA分离成多核糖体、单核糖体和无核糖体组分。使用称为“分子信标”的新型杂交探针和标准狭缝印迹法来检测在5'端附近含有连续低使用频率AGG（精氨酸）或同义高使用频率CGU插入的测试信使RNA。结果表明，与存在高使用频率密码子时相比，当信使RNA中存在低使用频率密码子时，游离信使RNA与结合信使RNA的比例增加了约两倍。相比之下，当插入连续低使用频率CUA或高使用频率CUG（亮氨酸）密码子，或者精氨酸密码子插入3'端附近时，游离信使RNA与结合信使RNA的比例没有差异。这些数据表明，在5'端翻译阻断过程中，至少一些信使RNA会因精氨酸密码子而非亮氨酸密码子从核糖体上释放，并且支持了在某些体内条件下，在没有终止密码子的情况下翻译可能会过早终止的观点。
