Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli.
Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli.
复制标题
核糖体准确性的提高会增加大肠杆菌中的程序化翻译移码。
DOI：
10.1073/pnas.90.6.2315
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发表时间：
1993
影响因子：
11.1
通讯作者：
Goldman,E
中科院分区：
文献类型：
--
作者：
Sipley,J;Goldman,E
我们测试了核糖体保真度增加对程序性释放因子2（RF 2）翻译移码的修改版本的影响。在测试的构建体中，移位位点处的原始UGA密码子被两个有义密码子中的任一个替换，UGG（色氨酸），其允许约13%的移码，或CUG（亮氨酸），其仅允许约2%的移码。我们证实了Curran和Yarus的结果[Curran，J.F. & Yarus，M.等人（1989）J. Mol. Biol.209，65-77]，包括在从tRNA基因的质粒拷贝诱导tRNA（Trp）后UGG移位的减少。但令我们惊讶的是，在一个高度精确的链霉素假依赖宿主中，UGG移码增加到50%以上。当我们向这些细胞中加入tRNA（Trp）质粒时，tRNA（Trp）基因的诱导将偏移降低到约7%。在这些不同的诱导条件下，信使RNA水平变化不大。其他增加的准确性等位基因也显示在移码位点与UGG的移码增加。所有增加的准确性等位基因导致更慢的翻译速率，并且框内报告基因的合成减少程度与框外报告基因的UGG移码程度之间似乎成比例。准确性的提高对较低水平的CUG移码几乎没有影响。然而，尽管tRNA（1 Leu）已经是大肠杆菌中最丰富的异源受体，但同源tRNA（1 Leu）的过量产生甚至显著降低了这种较低水平的移位。这些结果可以通过以下的假设合理化Curran和Yarus如下：与野生型核糖体，有限的可用性的tRNA（Trp）（约1%的总tRNA）促进暂停在UGG密码子（由于空缺的A位点），允许增加核糖体重新排列的机会。过量的tRNA（Trp）减少了A位点空缺的时间，从而减少了移码。较慢的超精确核糖体增加了停顿时间，从而增加了转移的机会，这一过程再次通过增加框内同源tRNA（Trp）而逆转。这些数据提供了强有力的支持，在一个程序化的移码位点的核糖体暂停时间的程度是一个主要的决定因素，移码的效率和tRNA的可用性可以是一个主要的影响这个过程的模型。
We have tested the effect of increased ribosomal fidelity on a modified version of the programmed release factor 2 (RF2) translational frameshift. In the constructs tested, the original UGA codon at the site of the shift was replaced by either of two sense codons, UGG (tryptophan), which allows a frameshift of approximately 13%, or CUG (leucine), which allows a frameshift of only approximately 2%. We confirmed the results of Curran and Yarus [Curran, J. F. & Yarus, M. (1989) J. Mol. Biol. 209, 65-77] in a wild-type ribosomal host, including a reduction of the UGG shift following induction of tRNA(Trp) from a plasmid copy of the tRNA gene. But to our surprise, in a hyperaccurate streptomycin pseudo-dependent host, the UGG frameshift increased to more than 50%. When we added a tRNA(Trp) plasmid to these cells, induction of the tRNA(Trp) gene reduced the shift back to approximately 7%. Messenger RNA levels did not vary greatly under these different induced conditions. Other increased accuracy alleles also showed increased frameshifting with UGG at the frameshift site. All increased accuracy alleles led to slower translation rates, and there appeared to be a proportionality between the extent of reduction of synthesis for the in-frame reporter and the extent of UGG frameshift for the out-of-frame reporter. There were little effects of increased accuracy on the lower level CUG frameshift. However, over-production of the cognate tRNA(1Leu) dramatically reduced even this lower level of shift, despite the fact that tRNA(1Leu) is already the most abundant isoacceptor in Escherichia coli. These results can be rationalized by following the hypothesis of Curran and Yarus as follows: with wild-type ribosomes, limited availability of tRNA(Trp) (about 1% of total tRNA) facilitates a pause at the UGG codon (due to the vacant A site), allowing increased opportunity for ribosome realignment. Excess tRNA(Trp) reduces the time the A site is vacant and thus reduces the frameshift. The slower hyperaccurate ribosomes increase the pause time and thus increase the opportunity for shifting, a process again reversed by increasing the in-frame cognate tRNA(Trp). These data provide strong support for a model in which the extent of ribosome pause time at a programmed frameshift site is a major determinant in the efficiency of the frameshift and in which tRNA availability can be a major influence on this process.