Lin Q, Yu N J, Spremulli L L
Department of Chemistry CB 3290, University of North Carolina, Chapel Hill 27599-3290, USA.
Plant Mol Biol. 1996 Dec;32(5):937-45. doi: 10.1007/BF00020490.
A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3chlM, molecular mass, 46 402) and the portion of this factor homologous to bacterial IF-3 (IF-3chlH, molecular mass 22 829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3chlM and IF-3chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3chlM and IF-3chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3chlH has at least 5-fold more activity than either native IF-3chl or IF-3chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3chl lying outside of the homology domain may down-regulate the activity of this factor.
一段预计编码纤细裸藻叶绿体翻译起始因子3成熟形式（IF-3chlM，分子量46402）的cDNA，以及该因子与细菌IF-3同源的部分（IF-3chlH，分子量22829）已被克隆，并作为组氨酸标签蛋白在大肠杆菌中表达。同源结构域能在大肠杆菌中以合理水平表达。然而，IF-3chlM毒性很强，只能少量产生。两种形式的叶绿体因子都与大肠杆菌核糖体相关。已开发出针对IF-3chlM和IF-3chlH的纯化程序，先用镍-亚氨基二乙酸（Ni-NTA）亲和层析，再用离子交换层析。IF-3chlM和IF-3chlH在促进核糖体解离以及促进甲酰甲硫氨酰-tRNA与大肠杆菌核糖体结合方面具有活性。然而，在携带天然翻译起始信号的mRNA存在的情况下，IF-3chlH在促进叶绿体30S核糖体亚基上起始复合物形成方面的活性比天然IF-3chl或IF-3chlM至少高5倍。这一观察结果表明，IF-3chl位于同源结构域之外的区域可能会下调该因子的活性。
