Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME*

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA^Gly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.     

nutR中的突变对λ噬菌体早期转录抗终止作用的影响

将λＤＮＡ的ｎｕｔＲ序列置于Ｌａｃ启动子的下游,在ｎｕｔＲ和β－半乳糖苷酶基因（ｇａｌＫ）之间插入λ噬茵体依赖ｒｈｏ的终止子ｔＲ１,使ｇａｌＫ的表达取决于Ｎ蛋白介导的转录抗终止作用。为研究ｎｕｔＲ对抗终止的影响,系统地进行了该序列中每个碱基的点突变（Ａ→Ｃ,Ｇ→Ｔ,Ｃ→Ａ及Ｔ→Ｇ）．结果表明ｂｏｘＡ中有两个碱基（位置２和５）的突变对抗终止作用是至关重要的,使抗终止效率降低了１０倍;而其他位置的改变影响甚微。ｂｏｘＡ的缺失在ｎｕｓ￣＋宿主中使抗终止数率降低了４０％,但在ｎｕｓＢ宿主中却恢复到野生型水平。ｂｏｘＢ茎环结构中,茎部顶端的碱基对及环中邻近茎部的两个碱基的突变对抗终止作用影响很大,被认为是Ｎ蛋白的识别序列．ｂｏｘＡ和ｂｏｘＢ之间的间隔序列中有两个碱基的突变几乎使抗终止作用丧失。     

λ噬菌体nutL序列突变对N蛋白生物功能的影响

λ噬菌体 Ｎ 蛋白不仅是抗转录终止的正调控因子,也是在翻译水平上阻遏自身基因表达的负调控蛋白．ｎｕｔ Ｒ Ｎ Ａ 位点（包括 ｂｏｘ Ａ 和 ｂｏｘ Ｂ）参与了这两种生物效应．对位于左向操纵子（ｐ Ｌ）的 ｎｕｔ Ｌ 序列进行了突变后,证明 ｎｕｔ Ｌ 缺失及 ｂｏｘ Ｂ 第 ６ 位核苷酸突变使 Ｎ 丧失了正、负调控功能,提示 ｎｕｔ Ｌ 在 Ｎ 介导的调控反应中是必需的．ｎｕｔ Ｌ 序列中 ｂｏｘ Ａ 单碱基突变使 Ｎ 丧失了正调控功能,部分保留了负调控功能．这种负调控作用导至极性效应,使 ｌａｃ Ｚ基因转录水平明显下降．     

Anisotropy studies of tRNA-T box antiterminator RNA complex in the presence of 1,4-disubstituted 1,2,3-triazoles

The binding of tRNA to the T box antiterminator RNA element is a critical component of the T box riboswitch mechanism that regulates essential genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened for disruption of the tRNA-T box antiterminator RNA interaction using a fluorescence anisotropy-based assay. Several compounds reduced the anisotropy greater than 50% likely indicating significant competition for binding antiterminator RNA. General structure-activity trends indicated that the substituents at both N-1 and C-4 likely are involved in ligand binding. In addition, the anisotropy of the complex was significantly decreased not only by ligands with the possibility for electrostatic interactions with the RNA, but also by ligands with the potential for π-π stacking or other hydrophobic interactions indicating that these non-electrostatic interactions could possibly be utilized in the future development of compounds that target and disrupt the function of this medicinally important riboswitch.