16S RNA在核糖体功能中的作用:单碱基改变及其对体外蛋白质合成的影响。

P R Cunningham, D Negre, C Weitzmann, R Denman, K Nurse, J Ofengand
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引用次数: 0

摘要

建立了大肠杆菌核糖体16S RNA位点特异性诱变的体外系统(Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987),在已知解码位点的残基C1400附近进行了10个单碱基改变。将C1400替换为U、A或G,在C1400两侧及两侧各缺失5个单碱基,并在C1400旁边插入C或U。另一个突变体在16S RNA的3'端拥有7个额外的核苷酸,这样就可以形成一个包含anti-Shine-Dalgarno序列的茎和环。另外一系列8个突变体测试了C1404、G1405和C1496、G1497之间假设的碱基配对。测定了这19个突变体在A和P位点结合以及起始依赖性和非起始依赖性肽合成中的活性。C1400的碱基置换均无明显抑制作用。插入和缺失完全阻断了起始依赖性肽的合成,但明显刺激了起始依赖性反应。tRNA结合的效果是可变的。唯一阻断所有核糖体功能的改变是G1401的缺失。3'端额外的茎和环阻断了起始依赖性肽的合成,但所有其他检测均正常。破坏和重组假设碱基对的突变体具有一种功能模式,表明相邻碱基对确实存在并且在功能上很重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
The role of 16S RNA in ribosome function: single base alterations and their effect on in vitro protein synthesis.

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site. C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed 7 additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Another series of 8 mutants tested hypothetical base-pairing between C1404, G1405 and C1496, G1497. The activity of these 19 mutants in A and P site binding, and in initiation-dependent and initiation-independent peptide synthesis was determined. None of the base substitutions of C1400 were strongly inhibitory. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects of tRNA binding were variable. The only alteration to block all ribosomal function was the deletion of G1401. The extra stem and loop at the 3'-end blocked initiation-dependent peptide synthesis, but all other assays were normal. The mutants which break and reform the hypothetical base-pairs had a functional pattern that suggest the contiguous base-pairs do exist and are functionally important.

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