Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNAMet and AUG selection
Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNAMet and AUG selection
复制标题
DOI：
10.1101/gad.1696608
复制
发表时间：
2008-08-15
影响因子：
10.5
通讯作者：
Hinnebusch, Alan G.
中科院分区：
文献类型：
--
作者：
Dong, Jinsheng;Nanda, Jagpreet S.;Hinnebusch, Alan G.
细菌70 S核糖体的高分辨率结构提供了mRNA和tRNA在延伸过程中与解码中心结合的原子细节，但这些信息缺乏前起始复合物（PIC）。我们确定了酵母18 S rRNA中的残基，这些残基在启动过程中对招募甲硫氨酰tRNAi（Met）至40 S亚基至关重要，通过分离降低GCN 4 mRNA翻译的突变。几个这样的Gcd（-）突变改变了螺旋28（h28）中的A928：U1389碱基对，并允许PIC扫描通常抑制GCN 4翻译的上游ORF的起始密码子。A928 U取代还损害体外重构系统中TC与PIC的结合。h28中凸起G926和某些其他残基的突变对应于与细菌70 S复合物中的P位点密码子或tRNA的直接接触，从而赋予Gcd（-）表型，该表型（如A928取代）通过过表达tRNAi Met而被抑制。因此，h28中的非保守的928：1389碱基对，加上对应于细菌核糖体中P位点接触的保守的18 S rRNA残基，对于真核生物中有效的Met-tRNAi（Met）结合和AUG选择是至关重要的。
High-resolution structures of bacterial 70S ribosomes have provided atomic details about mRNA and tRNA binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (PICs). We identified residues in yeast 18S rRNA critical in vivo for recruiting methionyl tRNAi(Met) to 40S subunits during initiation by isolating mutations that derepress GCN4 mRNA translation. Several such Gcd(-) mutations alter the A928:U1389 base pair in helix 28 (h28) and allow PICs to scan through the start codons of upstream ORFs that normally repress GCN4 translation. The A928U substitution also impairs TC binding to PICs in a reconstituted system in vitro. Mutation of the bulge G926 in h28 and certain other residues corresponding to direct contacts with the P-site codon or tRNA in bacterial 70S complexes confer Gcd(-) phenotypes that (like A928 substitutions) are suppressed by overexpressing tRNAi Met. Hence, the nonconserved 928: 1389 base pair in h28, plus conserved 18S rRNA residues corresponding to P-site contacts in bacterial ribosomes, are critical for efficient Met-tRNAi(Met) binding and AUG selection in eukaryotes.