Rossi P, de Crombrugghe B
Department of Genetics, University of Texas M.D. Anderson Hospital and Tumor Institute, Houston 77030.
Nucleic Acids Res. 1987 Nov 11;15(21):8935-56. doi: 10.1093/nar/15.21.8935.
A symmetrical sequence around the translation initiation site of several collagen genes is highly conserved. Deletions in this sequence increase translational efficiency of an alpha 2(I) collagen - CAT chimeric gene after DNA transfection of NIH 3T3 fibroblasts (Schmidt, Rossi and de Crombrugghe, submitted). The secondary structure, predicted by the sequence of this segment, was shown to exist in solution in 200 mM NaCl at 37 degrees C. Cell-free translation of the corresponding RNA using a reticulocyte lysate is inhibited 2 to 4-fold by preincubation with a 0.5 M NaCl extract of an NIH 3T3 ribosomal eluate. Cell-free translation of two mutant RNAs, with partial deletions of the conserved sequence, is not inhibited by such preincubation. This inhibition is not due to degradation of the RNA and requires a protein component of the ribosomal eluate, which, however, is not required after the preincubation step. Preincubation of the RNA with the ribosomal eluate from NIH 3T3 fibroblasts causes the reversible formation of an intermolecular dimer in which the conserved symmetrical sequences hybridize to each other. This results in an increase in the degree of secondary structure of the conserved segment around the translation initiation site. We speculate that translational efficiency could be modulated by influencing the equilibrium between monomer and dimer.
几个胶原蛋白基因翻译起始位点周围的对称序列高度保守。在将该序列缺失后，经DNA转染NIH 3T3成纤维细胞后，α2（I）胶原蛋白-CAT嵌合基因的翻译效率增加（施密特、罗西和德克伦布吕赫，已投稿）。根据该片段序列预测的二级结构，在37℃的200mM NaCl溶液中被证明存在。使用网织红细胞裂解物对相应RNA进行无细胞翻译时，与NIH 3T3核糖体洗脱液的0.5M NaCl提取物预孵育会抑制2至4倍。两个具有保守序列部分缺失的突变RNA的无细胞翻译不受这种预孵育的抑制。这种抑制不是由于RNA的降解，并且需要核糖体洗脱液的一种蛋白质成分，然而，在预孵育步骤之后则不需要该成分。将RNA与NIH 3T3成纤维细胞的核糖体洗脱液预孵育会导致分子间二聚体的可逆形成，其中保守的对称序列相互杂交。这导致翻译起始位点周围保守片段的二级结构程度增加。我们推测翻译效率可以通过影响单体和二聚体之间的平衡来调节。
