Valadez J G, Hernández-Sánchez J, Magos M A, Ontiveros C, Guarneros G
Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Mexico City, Mexico.
Mol Microbiol. 2001 Jan;39(2):361-9. doi: 10.1046/j.1365-2958.2001.02214.x.
Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth-defective cells. Two of these wild-type bar regions, barI+ and barII+, contain minigenes with similar AUG-AUA-stop codon sequences preceded by different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a result of deficiency of specific tRNA.
噬菌体λ无法在肽基 - tRNA水解酶（Pth）活性有缺陷的大肠杆菌突变体上进行营养生长。能使噬菌体在缺陷宿主上生长的突变位于λ基因组中名为bar的区域。从质粒构建体中表达野生型bar区域会导致蛋白质合成受到抑制，并对Pth缺陷细胞产生致死性。其中两个野生型bar区域，barI +和barII +，包含小基因，其具有相似的AUG - AUA - 终止密码子序列，前面有不同的Shine - Dalgarno（SD）序列和间隔区。从质粒构建体中诱导表达barI +和barII +区域会导致相似的蛋白质合成抑制模式和细胞生长停滞。因此，这些有害影响可能源于包含小基因双密码子“开放阅读框”（ORF）的转录本的翻译。为了测试这种可能性，我们检测了barI小基因内点突变的影响。结果表明，SD序列和双密码子“ORF”序列内的碱基对替换影响了蛋白质合成和细胞生长抑制。此外，每个突变体中的mRNA稳定性都发生了改变。较高的mRNA稳定性与毒性更强的小基因相关。我们认为这种效应可能是由于特定tRNA缺乏导致核糖体在暂停复合物中对mRNA进行保护所致。
