Single atom modification (O→S) of tRNA confers ribosome binding
Single atom modification (O→S) of tRNA confers ribosome binding
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DOI：
10.1017/s1355838299981529
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发表时间：
1999-02-01
期刊：
影响因子：
4.5
通讯作者：
Agris, PF
中科院分区：
文献类型：
--
作者：
Ashraf, SS;Sochacka, E;Agris, PF
大肠杆菌tRNA（SUU）（Lys）以及人tRNA（SUU）（Lys 3）在摆动位置34（s（2）U*（34））处具有2-硫尿苷衍生物。与天然tRNA（SUU）（Lys）不同，人tRNA（UUU）（Lys 3）的全长未修饰转录物和未修饰tRNA（UUU）（Lys 3）反密码子茎/环（ASL（UUU）（Lys 3））不结合AAA或AAG编程的核糖体。相比之下，完全未修饰的酵母tRNAP（Phe）反密码子茎/环（ASL（GAA）（Phe））具有与天然酵母tRNA（GmAA）（Phe）（K-d = 103 +/- 19 nM）相似的亲和力（K-d = 136 +/- 49 nM）。我们发现，s（2）U（34）对U-34的单一位点特异性取代产生修饰的ASL（SUU）（Lys）足以恢复核糖体结合。修饰的ASL（SUU）（Lys）以与天然tRNA（SUU）（Lys）（K-d = 70 +/- 7 nM）相当的亲和力（K-d = 176 +/- 62 nM）结合核糖体。此外，在与核糖体的结合中，修饰的ASL（SUU）（Lys 3）产生与天然大肠杆菌tRNA（SUU）（Lys）、酵母tRNA（GmAA）（Phe）和未修饰的ASL（GAA）（Phe）相同的16 S P-位点tRNA足迹。未修饰的ASL（UUU）（Lys 3）根本没有足迹。通过紫外光谱和核磁共振对其结构和热稳定性的研究表明，修饰的ASL（SUU）（Lys 3）环的动态构象与未修饰的ASL（UUU）（Lys）不同，而茎是同晶的。基于这些和其他数据，我们得出结论，tRNA（SUU）（Lys）和其他含s（2）u（34）的tRNA中的s（2）U（34）对于产生反密码子构象至关重要，该构象导致所有生物体中Po有效密码子相互作用。这是第一个单原子取代（U-34 -->（SU 34）-U-2）的例子，它赋予了核糖体结合在其他无活性tRNA上的特性。
Escherichia coli tRNA(SUU)(Lys), as well as human tRNA(SUU)(Lys3), has 2-thiouridine derivatives at wobble position 34 (s(2)U*(34)). Unlike the native tRNA(SUU)(Lys), the full-length, unmodified transcript of human tRNA(UUU)(Lys3) and the unmodified tRNA(UUU)(Lys3) anticodon stem/loop (ASL(UUU)(Lys3)) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAP(Phe) anticodon stem/loop (ASL(GAA)(Phe)) had an affinity (K-d = 136 +/- 49 nM) similar to that of native yeast tRNA(GmAA)(Phe) (K-d = 103 +/- 19 nM). We have found that the single, site-specific substitution of s(2)U(34) for U-34 to produce the modified ASL(SUU)(Lys) was sufficient to restore ribosomal binding. The modified ASL(SUU)(Lys) bound the ribosome with an affinity (K-d = 176 +/- 62 nM) comparable to that of native tRNA(SUU)(Lys) (K-d = 70 +/- 7 nM). Furthermore, in binding to the ribosome, the modified ASL(SUU)(Lys3) produced the same 16S P-site tRNA footprint as did native E, coli tRNA(SUU)(Lys), yeast tRNA(GmAA)(Phe), and the unmodified ASL(GAA)(Phe). The unmodified ASL(UUU)(Lys3) had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASL(SUU)(Lys3) was different from that of the unmodified ASL(UUU)(Lys), whereas the stems were isomorphous. Based on these and other data, we conclude that s(2)U(34) in tRNA(SUU)(Lys) and in other s(2)u(34)-containing tRNAs is critical for generating an anticodon conformation that leads Po effective codon interaction in all organisms. This is the first example of a single atom substitution (U-34 --> (SU34)-U-2) that confers the property of ribosomal binding on an otherwise inactive tRNA.