CONCERTED ACTIVITIES OF THE RNA RECOGNITION AND THE GLYCINE-RICH C-TERMINAL DOMAINS OF NUCLEOLIN ARE REQUIRED FOR EFFICIENT COMPLEX-FORMATION WITH PRERIBOSOMAL RNA
CONCERTED ACTIVITIES OF THE RNA RECOGNITION AND THE GLYCINE-RICH C-TERMINAL DOMAINS OF NUCLEOLIN ARE REQUIRED FOR EFFICIENT COMPLEX-FORMATION WITH PRERIBOSOMAL RNA
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DOI：
10.1111/j.1432-1033.1992.tb17318.x
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发表时间：
1992-10-15
期刊：
影响因子：
--
通讯作者：
ERARD, M
中科院分区：
文献类型：
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作者：
GHISOLFI, L;KHARRAT, A;ERARD, M
核仁蛋白是一种丰富的核仁蛋白，参与核糖体组装的早期阶段。核仁素的中央 40 kDa 结构域包含四个 RNA 识别基序 (RRM)，推测这些基序参与与前 rRNA 的特异性相互作用。为了详细检查该中心结构域的作用以及核仁素的N端和C端结构域对RNA结合的贡献，我们使用大肠杆菌表达系统合成了与这三个结构域及其子结构域的各种组合相对应的多肽。通过体外结合测定和对应于 pre-rRNA 中特定识别位点的合成 RNA，我们已经能够最终证明中央 40-kDa 结构域确实负责 RNA 识别的特异性，并且可以去除 N 端结构域而不影响 RNA 结合。最有趣的是，富含甘氨酸和精氨酸残基的 C 端 10 kDa 结构域对于核仁素与 RNA 的有效结合至关重要，但其本身并不有助于相互作用的特异性。 RNA 成分的圆二向色光谱探测表明，C 末端结构域显着改变了中央 RRM 核心的 RNA 结合特性。最后，红外光谱研究表明，中心 40 kDa 结构域由 α 螺旋和 β 片层构成，与特定前 rRNA 位点的相互作用会引起 β 片层构象的微妙变化。
Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.