Rheinberger H J, Sternbach H, Nierhaus K H
Proc Natl Acad Sci U S A. 1981 Sep;78(9):5310-4. doi: 10.1073/pnas.78.9.5310.
The binding of N-acetyl-Phe-tRNAPhe (an analogue of peptidyl-tRNA), Phe-tRNAPhe, and deacylated tRNAPhe to poly(U)-programmed tightly coupled 70S ribosomes was studied. The N-acetyl-Phe-tRNAPhe binding is governed by an exclusion principle: not more than one N-acetyl-Phe-tRNAPhe can be bound per ribosome, although this peptidyl-tRNA analogue can be present either at the aminoacyl-tRNA (A) site or the peptidyl-tRNA (P) site. Two Phe-tRNAPhe molecules are accepted by one ribosome in the presence of poly(U). This aminoacyl-tRNA binds enzymatically (in the presence of elongation factor Tu and GTP) and nonenzymatically to the A site and is then transferred to the P site, if that site is free. If this elongation factor G-independent movement is hampered, either by using an incubation temperature of 0 degrees C or by the addition of the translocation inhibitor viomycin, only one Phe-tRNAPhe per ribosome can be bound. The effect of the peptidyltransferase inhibitor chloramphenicol on the binding is similar to that of viomycin. In the absence of poly(U), Phe-tRNAPhe cannot bind to the ribosome. Deacylated [14C]tRNAPhe can bind in three copies to one ribosome. The new third tRNA binding site is called the "E" site. The sequence of filling the sites is P, E, and A. The apparent binding constants for the P and the E sites are both approximately 9 X 10(6) M-1 and that for the A site is 1.3 X 10(6) M-1. In the absence of poly(U), only one deacylated tRNAPhe can be bound per ribosome. This tRNAPhe most likely occupies the P site.
研究了N-乙酰苯丙氨酰-tRNAphe（肽基-tRNA的类似物）、苯丙氨酰-tRNAphe和脱酰基tRNAphe与聚尿苷酸编程的紧密偶联70S核糖体的结合情况。N-乙酰苯丙氨酰-tRNAphe的结合受排斥原理支配：每个核糖体结合的N-乙酰苯丙氨酰-tRNAphe不超过一个，尽管这种肽基-tRNA类似物可以存在于氨酰-tRNA（A）位点或肽基-tRNA（P）位点。在聚尿苷酸存在的情况下，一个核糖体可接纳两个苯丙氨酰-tRNAphe分子。这种氨酰-tRNA通过酶促作用（在延伸因子Tu和GTP存在下）和非酶促作用结合到A位点，然后如果P位点空闲，就转移到P位点。如果这种不依赖延伸因子G的移动受到阻碍，要么通过使用0℃的孵育温度，要么通过添加转位抑制剂紫霉素，每个核糖体只能结合一个苯丙氨酰-tRNAphe。肽基转移酶抑制剂氯霉素对结合的影响与紫霉素类似。在没有聚尿苷酸的情况下，苯丙氨酰-tRNAphe不能结合到核糖体上。脱酰基的[14C]tRNAphe可以以三个拷贝结合到一个核糖体上。新的第三个tRNA结合位点称为“E”位点。位点填充顺序为P、E和A。P位点和E位点的表观结合常数均约为9×10(6)M-1，A位点的表观结合常数为1.3×10(6)M-1。在没有聚尿苷酸的情况下，每个核糖体只能结合一个脱酰基tRNAphe。这种tRNAphe很可能占据P位点。
