Hüttenhofer A, Noller H F
Sinsheimer Laboratories, University of California at Santa Cruz 95064.
EMBO J. 1994 Aug 15;13(16):3892-901. doi: 10.1002/j.1460-2075.1994.tb06700.x.
We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.
我们使用针对mRNA糖磷酸骨架或碱基的化学探针，在有或没有tRNA（fMet）或tRNA（Phe）存在的情况下，对模型mRNA与30S核糖体亚基的相互作用进行了足迹分析。当在tRNA（fMet）存在下与30S亚基结合时，基因32 mRNA和022 mRNA的糖磷酸骨架在由位置-35（±2）和+19界定的约54个核苷酸区域内受到保护，免受羟基自由基攻击，当使用tRNA（Phe）时，延伸至位置+22。在70S核糖体中，保护在5'方向上延伸至约位置-39（±2）。在没有tRNA的情况下，30S亚基仅保护核苷酸-35（±2）至+5。在基因32 mRNA的位置+10和+11之间引入稳定的四环发夹结构不会干扰mRNA与30S亚基的tRNA（fMet）依赖性结合，但会导致位置+5下游mRNA糖磷酸骨架的保护丧失。使用碱基特异性探针，我们发现基因32 mRNA的Shine-Dalgarno序列（A-12、A-11、G-10和G-9）和起始密码子（A+1、U+2和G+3）在起始tRNA存在下受到30S亚基的强烈保护。在tRNA（Phe）存在下，相同的Shine-Dalgarno碱基受到保护，苯丙氨酸密码子的U+4、U+5和U+6也受到保护。有趣的是，起始密码子之前紧邻的A-1在与30S亚基和起始tRNA形成的复合物中受到保护，而在没有起始tRNA的情况下，U+2和G+3在与tRNA（Phe）形成的复合物中受到保护。此外，在任何一种tRNA存在下，Shine-Dalgarno区域上游的特定碱基（U-33、G-32和U-22）以及起始密码子3'端的碱基（G+11）都受到30S亚基的保护。这些结果表明，30S亚基的mRNA结合位点覆盖约54-57个核苷酸，并且与核糖体沿着其糖磷酸骨架与mRNA相互作用的可能性一致。
