Lancaster Lorna E, Savelsbergh Andreas, Kleanthous Colin, Wintermeyer Wolfgang, Rodnina Marina V
Institute of Molecular Biology, University of Witten/Herdecke, 58448 Witten, Germany.
Mol Microbiol. 2008 Jul;69(2):390-401. doi: 10.1111/j.1365-2958.2008.06283.x.
The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.
细胞毒素大肠杆菌素E3作用于细菌核糖体的30S亚基，并特异性地在解码中心切割16S rRNA，从而抑制翻译。尽管切割位点已为人所知，但尚不清楚翻译的哪个步骤受到抑制。我们通过分析蛋白质合成的各个步骤，研究了大肠杆菌素E3切割对核糖体功能的影响。我们发现，切割主要影响延伸步骤。大肠杆菌素E3切割的抑制作用源于连续受损解码事件的积累，每个事件都会导致A位点的占有率较低，从而降低延伸肽的产量。这种积累导致在读取几个密码子后翻译几乎完全停止。16S rRNA的切割不会损害在延伸因子Tu依赖的氨酰-tRNA结合过程中对密码子-反密码子复合物的监测或GTP酶激活，但会降低密码子识别复合物的稳定性，并减缓氨酰-tRNA在A位点的容纳。与完整核糖体相比，在大肠杆菌素E3切割的核糖体上，tRNA-mRNA易位更快，并且对抗生素紫霉素的抑制作用不太敏感。
