S4-ALPHA MESSENGER-RNA TRANSLATION REPRESSION COMPLEX .1. THERMODYNAMICS OF FORMATION
S4-ALPHA MESSENGER-RNA TRANSLATION REPRESSION COMPLEX .1. THERMODYNAMICS OF FORMATION
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DOI：
10.1016/0022-2836(87)90692-9
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发表时间：
1987-07-20
影响因子：
5.6
通讯作者：
THOMAS, MS
中科院分区：
文献类型：
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作者：
DECKMAN, IC;DRAPER, DE;THOMAS, MS
大肠杆菌四种核糖体蛋白的表达。操纵子（S4, S11， S13和L17）在翻译水平上通过与。α的结合受到调控。信使rna。使用过滤器结合试验和。α。通过体外转录制备的mRNA序列，先前的工作将S4靶位点定位在。apprx中。100个碱基的先导序列。我们已经扩展了这项工作，包括。alpha的片段。领导者有6个不同的5”终点和4个不同的3”终点。一个位于碱基23和69之间的核心区域（从大肠杆菌转录本的第一个核苷酸开始编号）以.apprx的亲和力结合S4。2 .mu.M-1。弱相互作用区域位于该核心的22个核苷酸5′和70个核苷酸3′；它们增加了S4对.apprx的亲和力。13 .mu.M-1。S4- α的研究。mRNA在不同条件下的结合揭示了以下情况。(1)特异性和非特异性结合对K+浓度的依赖性相同，均为。vdelta。日志K / .vdelta。log [K+] .apprxeq。4在大多数钾盐中。对于KCl和KBr，观察到特异性复合物形成的盐依赖性要弱得多，这表明蛋白质通过结合卤化物阴离子来响应正确的RNA底物。(2)在1 ~ 4mm之间增加MgCl2的浓度，使其结合能力提高4倍，在20mm以内没有进一步的影响。复合物吸收了约5个Mg2+，平均结合常数为。apprx。每台600 M-1。在MgCl2的存在下，RNA的恢复也是获得完全结合所必需的。这些效果只能在。alpha中看到。超出起始密码子的mRNA；S4和。结合。前导序列本身对Mg2+不敏感。(3)结合动力学快速且可能受扩散控制。(4)络合物的形成完全是熵驱动的。
Expression of the four ribosomal proteins from the Escherichia coli .alpha. operon (S4, S11, S13, and L17) is regulated at the level of translation by the binding of S4 to the .alpha. mRNA. Using a filter binding assay and .alpha. mRNA sequences prepared by in-vitro transcription, previous work located the S4 target site within the .apprx. 100-base leader sequence. We have extended this work to include fragments of the .alpha. leader with six different 5'' end points and four different 3'' end points. A core region between bases 23 and 69 (numbering from the first nucleotide of the E. coli transcript) binds S4 with an affinity of .apprx. 2 .mu.M-1. Regions of weak interactions are located in the 22 nucleotides 5'' and the 70 nucleotides 3'' to this core; they increase the S4 affinity to .apprx. 13 .mu.M-1. Studies of S4-.alpha. mRNA binding under different conditions have revealed the following. (1) Specific and non-specific binding show the same dependence on K+ concentration, with .vdelta. log K/.vdelta. log [K+] .apprxeq. 4 in most potassium salts. With KCl and KBr, much weaker salt dependence of specific complex formation is observed suggesting that the protein responds to the correct RNA substrate by binding halide anions. (2) Increasing the MgCl2 concentration between 1 and 4 mM enhances binding by a factor of 4, with no further effects up to 20 mM. About five Mg2+ are taken up by the complex with an average binding constant of .apprx. 600 M-1 each. Renaturation of the RNA in the presence of MgCl2 is also required to obtain full binding. These effects are seen only with .alpha. mRNA extending beyond the initiation codon; S4 binding to the .alpha. leader sequence itself is insensitive to Mg2+. (3) The association kinetics are fast and probably diffusion controlled. (4) Formation of the complex is entirely entropy driven.