tRNA from E.coli/大肠杆菌tRNA

大肠杆菌tRNA的中文介绍  
大肠杆菌（*Escherichia coli*）的转运RNA（tRNA）是蛋白质合成的核心分子，负责将氨基酸精准传递至核糖体，并根据mRNA的密码子序列组装多肽链。以下从结构、加工、基因组织及功能调控等方面详述其特性：  

1. **分子结构与功能**  
- **二级结构**：tRNA呈典型的三叶草结构，包含四个功能区域：接受茎（3'端携带氨基酸）、D环、反密码子环（含反密码子）和TΨC环。反密码子环中的三核苷酸序列（反密码子）与mRNA上的密码子互补配对，确保氨基酸的精准定位。  
- **接受茎与氨基酰化**：接受茎的3'末端通过高能酯键连接特定氨基酸，这一过程由氨基酰-tRNA合成酶（AARS）催化。AARS分为I类和II类，分别将氨基酸连接到tRNA的2'-OH或3'-OH位，但溶液中两者可自发异构化。  

2. **tRNA的加工过程**  
- **前体转录与切割**：大肠杆菌的tRNA基因大多以多顺反子形式存在，与rRNA或蛋白质基因共转录。前体tRNA（pre-tRNA）需经历多步加工：  
  1. **5'端切割**：由核酶RNase P（含RNA亚基M1和蛋白质C5）切除5'前导序列。  
  2. **3'端修剪**：通过内切酶（如RNase E）和外切酶（如RNase T）逐步切除3'尾随序列，最终暴露或添加CCA序列。约30%的tRNA需通过tRNA核苷酸转移酶在3'端后加CCA，该酶无需模板即可依次添加C、C、A。  
- **碱基修饰**：包括甲基化、硫代化等，增强tRNA稳定性和功能特异性。  

3. **基因组织与表达调控**  
- **基因分布**：大肠杆菌基因组含86个tRNA基因，编码40种成熟tRNA异型（对应20种氨基酸）。其中64个基因位于多顺反子转录单元，常与rRNA或蛋白基因共存；其余为单顺反子。  
- **动态调控**：在氨基酸饥饿条件下，tRNA的稳定性显著下降，20分钟内大部分被降解，以快速响应代谢压力。此外，多腺苷酸化酶（PAP I）通过在前体tRNA的3'端添加短腺苷酸尾，与外切酶竞争，调控tRNA的成熟与稳定性。  

4. **研究案例与技术进展**  
- **亮氨酸tRNA（tRNA^Leu^）** ：通过优化提取与纯化方法，研究者获得高纯度tRNA^Leu^，其3'和5'端存在额外核苷酸，且能特异性结合亮氨酸。  
- **基因工程应用**：利用质粒pTrc99B-Leu1/Leu2在大肠杆菌中高效表达tRNA^Leu^，为研究tRNA与合成酶的相互作用提供材料。  

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Introduction to *Escherichia coli* tRNA (English Version)  
The transfer RNA (tRNA) of *Escherichia coli* serves as a molecular adaptor that links mRNA codons to amino acids during protein synthesis. Below is a comprehensive overview of its structure, biogenesis, and functional regulation:  

1. **Molecular Architecture and Functional Domains**  
- **Secondary Structure**: tRNA adopts a cloverleaf-like fold with four domains: the acceptor stem (3' end for amino acid attachment), D-loop, anticodon loop (containing the anticodon triplet), and TΨC loop. The anticodon pairs with mRNA codons via complementary base pairing.  
- **Aminoacylation**: Aminoacyl-tRNA synthetases (AARSs) catalyze the attachment of specific amino acids to the 3'-OH or 2'-OH of the terminal ribose in the acceptor stem. Class I and II AARSs differ in their binding modes, but spontaneous transacylation between 2' and 3' isomers occurs in solution.  

2. **tRNA Maturation and Processing**  
- **Precursor Cleavage**: Most tRNA genes are transcribed as polycistronic units alongside rRNA or protein-coding genes. Key processing steps include:  
  1. **5' End Trimming**: Catalyzed by RNase P, a ribozyme composed of an RNA subunit (M1) and a protein subunit (C5).  
  2. **3' End Maturation**: Achieved via endonucleases (e.g., RNase E) and exonucleases (e.g., RNase T). For some tRNAs, the 3' CCA sequence is added post-transcriptionally by tRNA nucleotidyltransferase, which sequentially incorporates CTP, CTP, and ATP without a template.  
- **Nucleotide Modifications**: Chemical modifications (e.g., methylation, thiolation) enhance tRNA stability and decoding accuracy.  

3. **Genomic Organization and Regulatory Mechanisms**  
- **Gene Clusters**: The *E. coli* genome harbors 86 tRNA genes encoding 40 mature isoacceptors. Approximately 75% reside in polycistronic operons, while others are monocistronic.  
- **Dynamic Regulation**: Under amino acid starvation, tRNAs undergo rapid degradation within 20 minutes, reflecting their role in metabolic adaptation. Polyadenylation by PAP I competes with 3'→5' exonucleases, modulating tRNA stability and functional availability.  

4. **Research Advances and Applications**  
- **Leucine tRNA (tRNA^Leu^)**: High-purity tRNA^Leu^ was isolated with unique terminal nucleotides, demonstrating specific leucine-binding capacity.  
- **Genetic Engineering**: Plasmid-based overexpression systems (e.g., pTrc99B-Leu1/Leu2) enable large-scale production of tRNA^Leu^, facilitating studies on tRNA-synthetase interactions.  

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