The polypeptide chain release factor eRF1 specifically contacts the s4UGA stop codon located in the A site of eukaryotic ribosomes
The polypeptide chain release factor eRF1 specifically contacts the s4UGA stop codon located in the A site of eukaryotic ribosomes
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DOI：
10.1046/j.1432-1327.2001.02177.x
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发表时间：
2001-05-01
期刊：
影响因子：
--
通讯作者：
Favre, A
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文献类型：
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作者：
Chavatte, L;Frolova, L;Favre, A
它以前已经被证明[布朗，C. M。和Tate，W.P.（1994）J.Biol.Chem.269，33164-33170.]在原核生物中参与翻译终止的多肽链释放因子RF 2能够与含有终止信号的微型信使RNA发生光交叉反应，其中U被4-硫尿苷（s（4）U）取代。在这里，使用相同的策略，我们已经监测了在tRNA（f）（Met）存在下的14-mer mRNA和在tRNA（Asp）（tRNA（Asp）基因转录物）存在下的42-mer mRNA与真核核糖体组分的光交联。我们发现：（a）14-mer和42-mer mRNA都与核糖体RNA和核糖体蛋白发生交叉反应。交联的核糖体蛋白的模式与两种mRNA相似，并且对离子条件敏感;（B）用42-mer mRNA获得的交联模式在加入tRNA（Asp）后显示出特征性修饰，为适当的mRNA定相到核糖体上提供了证据。（c）当真核多肽链释放因子1（eRF 1）加入核糖体tRNA（Asp）复合物中时，它与含有位于A位点的s（4）UGA终止密码子的42-mer mRNA发生交叉反应，但不与s（4）UCA正义密码子发生交叉反应;这种交联涉及eRF 1的N-末端和中间结构域，但不涉及与真核多肽链释放因子3（eRF 3）相互作用的C结构域;（d）eRF 3的添加对eRF 1 -42-mer mRNA交联的产率没有影响，并且eRF 3不与42-mer mRNA交叉反应。这些实验描述了允许真核核糖体上mRNA最佳定相的体外条件，并证明了“核心”eRF 1和s（4）UGA终止密码子在核糖体A位点内的直接和特异性接触。
It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem. 269, 33164-33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s(4)U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of tRNA(f)(Met), and 42-mer mRNA in the presence of tRNA(Asp) (tRNA(Asp) gene transcript). We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNA(Asp) providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14-mer mRNA.tRNA(f)(Met) pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome.tRNA(Asp) complex it crossreacts with the 42-mer mRNA containing the s(4)UGA stop codon located in the A site, but not with the s(4)UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1-42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of 'core' eRF1 and s(4)UGA stop codon within the ribosomal A site.