Binding of L7Ae protein to the K-turn of archaeal snoRNAs:: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea
Binding of L7Ae protein to the K-turn of archaeal snoRNAs:: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea
复制标题
DOI：
10.1093/nar/gkg175
复制
发表时间：
2003-02-01
影响因子：
14.9
通讯作者：
Hüttenhofer, A
中科院分区：
文献类型：
--
作者：
Rozhdestvensky, TS;Tang, TH;Hüttenhofer, A
核仁小RNA（在真核生物中称为snoRNA，在真核生物中称为sRNA）可以分为H/ACA或C/D盒snoRNA（sRNA）亚类。在真核生物中，H/ACA snoRNA组装成核糖核蛋白（RNP）复合物，该复合物包含四种蛋白质：Cbf 5 p，Gar 1 p，Nop 10 p和Nhp 2 p。到目前为止，在古细菌H/ACA RNP中尚未鉴定出Nhp 2 p蛋白的同源物，而其他三种蛋白质的潜在直系同源物已经被鉴定。Nhp 2 p与古细菌核糖体蛋白和C/D盒蛋白L7 Ae相关，特别是在蛋白质序列的中间部分。这一发现表明L7 Ae可能能够取代古细菌H/ACA sRNA中的Nhp 2 p蛋白。通过带移分析，我们在体外分析了H/ACA盒sRNA与古生菌闪烁古生菌蛋白L7 Ae之间的相互作用。我们提出的证据表明，L7 Ae形成特定的复合物与三种不同的H/ACA sRNA，命名为Afu-4，Afu-46和Afu-190与表观Kd范围从28到100 nM。通过化学和酶的探测，我们发现，不同的基地内的H/ACA sRNAs的凸起或环与L7 Ae蛋白相互作用。这些发现证实了L7 Ae结合位点的突变分析。因此，L7 Ae结合所需的RNA基序表现出被称为K-转角的结构，其存在于所有C/D盒sRNA中。我们还确定了四个H/ACA RNA的古细菌物种Pyrococcus表现出的K-转折基序在其结构中的类似位置。这些发现表明L7 Ae蛋白在大肠杆菌中的三重作用，例如在核糖体以及H/ACA和C/D盒sRNP生物发生中，并通过与K-转角基序结合而发挥功能。
Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent K-d ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.