Allen P N, Noller H F
Thimann Laboratories, University of California, Santa Cruz 95064.
J Mol Biol. 1989 Aug 5;208(3):457-68. doi: 10.1016/0022-2836(89)90509-3.
We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.
我们使用一组对结构敏感的化学探针，研究了蛋白质突变对大肠杆菌核糖体中16S rRNA高级结构的影响。研究了十个突变菌株，这些菌株的核糖体蛋白S4和S12发生了改变，包括同时含有改变的S4和S12的双突变体。两个核糖体模糊（ram）S4突变菌株、四个链霉素抗性（SmR）S12突变菌株、一个链霉素假依赖（SmP）S12突变菌株、一个链霉素依赖（SmD）S12突变菌株和两个链霉素独立（Sm1）双突变体（同时含有SmD和ram突变）接受了探测，并与同基因野生型菌株进行了比较。在含有S4 ram突变的菌株的核糖体中，核苷酸A8和A26在其N-1位置对硫酸二甲酯（DMS）的反应性增强。在携带SmD等位基因的菌株的核糖体中，A908、A909、A1413和G1487对化学探针的反应性显著降低。当双突变体中同时存在S4和S12突变时，也观察到了相同的效应。发现A908的反应性与SmR、SmD、SmP和野生型核糖体的错义编码潜力之间存在有趣的相关性；随着核糖体翻译错误频率的增加，A908的反应性增加。在ram核糖体的情况下，A908的反应性类似于野生型，除非tRNA结合，在这种情况下它会变得高反应性。同样，链霉素对野生型核糖体中的A908影响很小，除非tRNA结合，在这种情况下其反应性增加，类似于结合了tRNA的ram核糖体。最后，链霉素与SmP和SmD核糖体的相互作用导致A908的反应性增加到接近野生型水平。提出了一个简单的模型，其中A908的反应性反映了30S亚基两种构象状态之间平衡的位置，其中一种对DMS有反应性，另一种对DMS无反应性。在这个模型中，这两种状态之间的平衡将受蛋白质S4和S12的影响。S12中的突变通常会导致向无反应性构象的转变，在SmD和SmP核糖体的情况下，这种转变可以被链霉素表型抑制，蛋白质S4中的ram突变会导致向有反应性构象的转变，但仅在tRNA结合时。这表明这两类突变的相反作用通过 somewhat 不同的机制影响校对过程。
