Ericson G, Minchew P, Wollenzien P
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical Center, MO 63104, USA.
J Mol Biol. 1995 Jul 21;250(4):407-19. doi: 10.1006/jmbi.1995.0386.
Escherichia coli 30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association. In the inactive form, 16 S rRNA base-pairs (921-922).(1395-1396) and (923-925).(1391-1393), which are part of region 28, are unstable and an alternate arrangement, (921-923).(1532-1534), is detected by psoralen photochemical crosslinking. Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit. 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit. On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions. When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929. Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926. Mutations were made to insert or delete a nucleotide at position 920. The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926. We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding. Molecular models of region 28 were made using the program MC-SYM. Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity.
在低单价或二价阳离子浓度下，大肠杆菌30 S核糖体亚基会发生可逆变化，并在tRNA结合和50 S亚基结合方面变得无活性。在无活性形式下，作为28区一部分的16 S rRNA碱基对（921 - 922）.（1395 - 1396）和（923 - 925）.（1391 - 1393）不稳定，通过补骨脂素光化学交联检测到一种替代排列（921 - 923）.（1532 - 1534）。定点诱变已被用于研究28区碱基配对区域的变化或替代二级结构是否是亚基无活性的原因。用C1533A替代或缺失核苷酸1534至1542的30 S亚基仍能像野生型30 S亚基一样被灭活。另一方面，在920至926区域含有序列变化的30 S亚基，即使在激活条件下，tRNA结合也会出现中度至重度下降。当对含有这些突变的30 S亚基进行化学探测时，它们未显示出核苷酸G926正常的高反应性，相反，反应性转移到了G925或G928以及G929。920区域的两个突变导致A1394碱基配对而不是像正常情况那样未配对的结构；缺失而不是替代A1394导致tRNA结合活性丧失和G926反应性降低。在920位进行了插入或缺失一个核苷酸的突变。缺失突变体而非插入突变体具有降低的tRNA结合活性以及G926的低反应性。我们得出结论，28区的结构变化解释了tRNA结合中活性/无活性的差异。使用程序MC - SYM构建了28区的分子模型。包含A1394和G1392之间氢键相互作用的模型解释了野生型序列中G926的反应性，并解释了大多数突变在改变G926反应性方面的影响。
