关于tRNA的误区及点拨

本文在高中生物学课本的基础上,进一步对tRNA作了多角度的介绍。     

Analysis of the 3′ ends of tRNA as the cause of insertion sites of foreign DNA in Prochlorococcus

The purpose of this study was to investigate the characteristics of transfer RNA (tRNA) responsible for the association between tRNA genes and genes of apparently foreign origin (genomic islands) in five high-light adapted Prochlorococcus strains. Both bidirectional best BLASTP (basic local alignment search tool for proteins) search and the conservation of gene order against each other were utilized to identify genomic islands, and 7 genomic islands were found to be immediately adjacent to tRNAs in Prochlorococcus marinus AS9601, 11 in P. marinus MIT9515, 8 in P. marinus MED4, 6 in P. marinus MIT9301, and 6 in P. marinus MIT9312. Monte Carlo simulation showed that tRNA genes are hotspots for the integration of genomic islands in Prochlorococcus strains. The tRNA genes associated with genomic islands showed the following characteristics: (1) the association was biased towards a specific subset of all iso-accepting tRNA genes; (2) the codon usages of genes within genomic islands appear to be unrelated to the codons recognized by associated tRNAs; and, (3) the majority of the 3′ ends of associated tRNAs lack CCA ends. These findings contradict previous hypotheses concerning the molecular basis for the frequent use of tRNA as the insertion site for foreign genetic materials. The analysis of a genomic island associated with a tRNA-Asn gene in P. marinus MIT9301 suggests that foreign genetic material is inserted into the host genomes by means of site-specific recombination, with the 3′ end of the tRNA as the target, and during the process, a direct repeat of the 3′ end sequence of a boundary tRNA (namely, a scar from the process of insertion) is formed elsewhere in the genomic island. Through the analysis of the sequences of these targets, it can be concluded that a region characterized by both high GC content and a palindromic structure is the preferred insertion site.     

大肠杆菌亮氨酰-tRNA合成酶与tRNALeu的相互作用头孢菌素酰化酶的表达、翻译后加工和亚基重组

第一部分:为得到大量的大肠杆菌tRNALeu1 和tRNALeu2 ,研究tRNA与亮氨酰 tRNA合成酶(LeuRS)的相互作用,用基因克隆的方法高表达并纯化了它们;测定了接受活力和被LeuRS催化的动力学常数;改进了RNA体外转录必需的T7RNA聚合酶的纯化方法,优化了转录条件,转录序列比文献值高 1 0倍.用体外转录方法得到了各种在接受茎突变的tRNALeu变种,研究了一个在CP1结构域插入 40个氨基酸残基的变种LeuRS A对tRNALeu2种等受体的识别,证明了tRNA Leu1 和tRNA Leu2 接受茎上的第一对硷基对是否摆动是LeuRS A识别的关键.　　第二部分:在大肠杆菌中高表达头孢菌素酰化酶基因 1 0倍以上.研究了它的α 亚基的N 端变化(LAEP →MGIP )对酶学动力学常数的影响.构建了带有不同抗菌素抗性和启动子的高表达质粒,研究了它们表达的特点,分析了这些质粒的不同用途.用含有编码该酶两个亚基的基因的相容质粒共转化的方法.研究了它们在体内的重组和前体的加工.研究结果表明,该酶可能属于一类新的N 端亲和水解的酰化酶