Analysis of initiation factor function in highly fractionated and unfractionated reticulocyte lysate systems.
Analysis of initiation factor function in highly fractionated and unfractionated reticulocyte lysate systems.
复制标题
高度分级和未分级网织红细胞裂解物系统中起始因子功能的分析。
DOI：
10.1016/s0076-6879(79)60008-3
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发表时间：
1979
影响因子：
--
通讯作者：
W. Kemper
中科院分区：
文献类型：
--
作者：
B. Safer;R. Jagus;W. Kemper
由于这些复合物的不稳定性，确定起始因子、tRNA、mRNA和起始前复合物的核苷酸组成以及未分离和重组体系中翻译组分的分布变得复杂。这导致了各种翻译组分的明显低效利用，以及在80s起始复合物组装过程中形成的中间体的形成和组成要求的不确定性。然而，由于翻译成分的人工关联很容易发生，因此使用交联剂来稳定这些配合物会带来额外的问题。我们发现，在蔗糖密度梯度分析过程中，将不可水解的GTP类似物GDPNP交换为核糖体结合的GTP，或者GTP再生系统的存在，可以在不使用化学固定的情况下高效稳定地结合RNA和起始因子。最近对标准蔗糖密度梯度技术、聚丙烯酰胺凝胶电泳分析和样品制备、氨基酰基trna和mRNA分析、真核起始因子制备的快速程序以及不改变大多数起始因子功能活性的放射性标记技术的改进，现在允许进行检查
Determination of the initiation factor, tRNA, mRNA, and nucleotide composition of preinitiation complexes and the distribution of translational components in both unfractionated and reconstituted systems has been complicated by the instability of these complexes. This has resulted in an apparent inefficient utilization of various translational components, as well as uncertainties in the requirements for formation and composition of intermediates formed during assembly of the 80 S initiation complex. Additional problems are introduced, however, by the use of cross-linking~: eagents to stabilize these complexes, since artificial associations of translational components can easily occur.We have found that exchange of the nonhydrolyzable GTP analog, GDPNP, for ribosomal bound GTP, or the presence of GTP-regenerating systems, permits highly efficient and stable binding of RNA and initiation factors during sucrose density gradient analysis without the use of chemical fixation. 1o Recent modifications in standard sucrose density gradient techniques, polyacrylamide gel electrophoretic analysis and sample preparation, aminoacyl-tRNA and mRNA analysis, rapid procedures for eukaryotic initiation factor preparation, and radiolabeling techniques which do not alter the functional activities of most initiation factors, now permit exam-