Li C, Reches M, Engelberg-Kulka H
Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
J Bacteriol. 2000 Nov;182(22):6302-7. doi: 10.1128/JB.182.22.6302-6307.2000.
The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.
通常作为终止密码子的UGA密码子，也能指导将氨基酸硒代半胱氨酸掺入蛋白质中。这种UGA解码过程需要一种称为硒代半胱氨酸插入序列（SECIS）的顺式作用mRNA元件，它能形成茎环结构。在大肠杆菌中，硒代半胱氨酸的掺入仅需要fdhF SECIS的17个核苷酸长的上部茎环结构。该结构在第17位带有一个凸起的核苷酸U。在这里，我们研究了位于大肠杆菌fdhF SECIS上部茎环结构中的单个凸起核苷酸是否参与了与SelB的体内相互作用。我们采用遗传学方法，生成并鉴定了抑制SECIS中凸起核苷酸突变的selB突变。分离出的所有selB抑制突变都聚集在SelB C端亚结构域4b中对应28个氨基酸的区域。还发现这些selB抑制突变能抑制大肠杆菌SECIS环部或上部茎部的突变。因此，大肠杆菌SECIS上部茎环结构可被视为一个“单一可抑制单元”，这表明该元件与SelB之间相互作用的性质具有一定的灵活性。
