Makhno V I, Peshin N N, Semenkov Iu P, Kirillov S V
Mol Biol (Mosk). 1988 May-Jun;22(3):670-9.
The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.
广泛存在的“紧密型”70S核糖体的功能活性通常为55%至80%。我们在此表明，这类核糖体的无活性部分实际上被残留的内源性RNA所阻断。通过在分离程序中增加一个步骤可去除这些RNA，该步骤包括：1. 在解离条件下，即在含有3 mM MgCl2和200 mM NH4Cl的缓冲液中，对“紧密型”70S进行短时间加热（15分钟，37摄氏度）；2. 在相同缓冲液的蔗糖密度梯度上洗去内源性RNA；3. 在含有5 mM MgCl2和50 mM NH4Cl的蔗糖梯度上最终筛选纯化的“紧密型”70S。通过这种程序分离的“紧密型”70S核糖体在tRNA结合（包括因子依赖性结合）、肽键合成和转位方面具有90%至100%的活性。
