Geigenmüller U, Hausner T P, Nierhaus K H
Eur J Biochem. 1986 Dec 15;161(3):715-21. doi: 10.1111/j.1432-1033.1986.tb10498.x.
The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.
用于确定结合的tRNA核糖体位点位置的标准技术，即嘌呤霉素反应，已就其在tRNA饱和条件下的适用性进行了分析。所推导的标准已用于重新审视肽基-tRNA结合的排除原则，该原则指出每个核糖体原则上有两个位点（A位点和P位点）可供使用，但只能结合一个肽基-tRNA（乙酰苯丙氨酰-tRNA）。得到了以下结果。如果反应条件能防止一个核糖体进行多次嘌呤霉素反应，那么嘌呤霉素反应才适合用于位点测定。当乙酰苯丙氨酰-tRNA相对于核糖体过量，且不存在延伸因子G（EF-G）时，在0℃满足该标准，此时结合在P位点的物质与嘌呤霉素反应（50小时后定量反应），而结合在A位点的物质则不反应。相反，在37℃时，嘌呤霉素反应的程度可能超过结合值2至4倍（“重复反应”）。在有EF-G存在的情况下，即使在0℃也会出现重复的嘌呤霉素反应，即EF-G在0℃时就能促进转位反应。然而，如果仅使用催化量的EF-G，在0℃短时间孵育（长达60分钟）时，转位程度会变得极低。采用上述标准，通过两种不同的实验策略证实了大肠杆菌核糖体排除原则的有效性。核糖体用每个70S核糖体一个分子的乙酰苯丙氨酰-tRNA饱和，定量的嘌呤霉素反应证明了乙酰苯丙氨酰-tRNA只位于P位点。在存在紫霉素（它会阻断转位反应）的情况下也得到了相同的结果。这些发现还表明，这里几乎100%的核糖体参与了乙酰苯丙氨酰-tRNA与P位点的结合。每个核糖体用一个乙酰-[14C]苯丙氨酰-tRNA分子预先装载70S核糖体的P位点，可防止额外的乙酰-[3H]苯丙氨酰-tRNA结合。相反，每个核糖体携带一个[14C]苯丙氨酰-tRNA分子的70S颗粒，每个核糖体最多还能结合0.64个分子的乙酰-[3H]苯丙氨酰-tRNA。
