Adenine-containing codons enhance protein synthesis by promoting mRNA binding to ribosomal 30S subunits provided that specific tRNAs are not exhausted
Adenine-containing codons enhance protein synthesis by promoting mRNA binding to ribosomal 30S subunits provided that specific tRNAs are not exhausted
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DOI：
10.1016/j.biochi.2011.09.019
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发表时间：
2012-03-01
期刊：
影响因子：
3.9
通讯作者：
Hernandez-Sanchez, J.
中科院分区：
文献类型：
--
作者：
Castillo-Mendez, M. A.;Jacinto-Loeza, E.;Hernandez-Sanchez, J.
起始密码子下游的腺嘌呤促进蛋白质合成；然而，当同源tRNA耗尽时，一些早期位置的含腺嘌呤密码子（AGA， AGG和AUA）会抑制蛋白质合成。虽然不令人信服，但也有报道称腺嘌呤的存在增强了mRNA与核糖体的结合。为了理解这些明显的不一致性，我们分析了这些密码子对mRNA-核糖体结合强度、mRNA稳定性、肽基trna （pep-tRNA）的产生和蛋白质合成的影响。通过位点定向诱变获得含有lacZ衍生物的构建体，将GGG、AGG、AGA、ATA和AAA密码子序列插入密码子2-3和3-4位置。在野生型转录-翻译系统中，与含有丰富鸟嘌呤的密码子（GGG和AGG）相比，含有更多腺嘌呤的密码子（AAA、ATA和AGA）比含有更多鸟嘌呤的密码子（GGG和AGG）更能促进β -半乳糖苷酶（β -gal）的合成。当富腺嘌呤构建体在野生型细胞中表达时，也检测到全长mrna。在trna池容易耗尽的条件下（pep-tRNA水解酶缺陷细胞），富含腺嘌呤的lacZ衍生物对蛋白质合成和细胞生长产生更强的抑制作用。除了ATA lacZ衍生物外，只有含有饥饿密码子的质粒构建物在Pth缺陷细胞中产生pep-tRNA （AGA和较小程度的AGG）。含有更多腺嘌呤的密码子明显促进了lacZ mRNA与30S亚基的结合。在结合实验前，通过加热破坏mRNA二级结构，GGG lacZ mRNA的结合程度有所增加，这与MFOLD预测的lacZ mRNA二级结构一致。总之，这些结果表明，无论密码子的使用如何，mRNA与核糖体的结合在增强富含腺嘌呤的密码子的翻译中起着重要作用。这种作用在野生型系统中自然表达，并取决于腺嘌呤含量，而在Pth缺陷细胞中过度表达含有罕见密码子的lacZ衍生物会产生抑制作用。(C) 2011 Elsevier Masson SAS。版权所有。
Adenines downstream of the initiation codon promote protein synthesis; however, some adenine-containing codons (AGA, AGG and AUA) at early positions inhibit protein synthesis when cognate tRNA is exhausted. It has also been reported, although not convincingly, the presence of adenines enhancing mRNA binding to the ribosome. To understand these apparent inconsistencies we analyzed the effect of these codons in mRNA-ribosome binding strength, mRNA stability, the production of peptidyl-tRNA (pep-tRNA) and protein synthesis. Constructs harboring lacZ derivatives were obtained by site directed mutagenesis where tandems of GGG, AGG, AGA, ATA and AAA codons were inserted at codon positions 2-3 and 3-4. Codons containing more adenines, irrespective of being common or rare, (AAA, ATA and AGA) promoted a higher synthesis of beta-galactosidase (beta-gal) in comparison with those rich in guanines (GGG and AGG) in a wild type transcription-translation system. Full-length mRNAs were also detected when the adenine-rich constructs were expressed in wild type cells. Under conditions where the pool of tRNAs is readily exhausted (pep-tRNA hydrolase defective cells), the adenine-rich lacZ derivatives caused a stronger and general inhibition of protein synthesis and cell growth. With the exception of the ATA lacZ derivative, only plasmid constructs containing hungry codons generated pep-tRNA (AGA and to a lesser extent AGG) in Pth defective cells. Codons containing more adenines clearly promoted lacZ mRNA binding to 30S subunit. The GGG lacZ mRNA showed a moderate increase in binding when mRNA secondary structures were disrupted by heating mRNAs before the binding assay which agrees with the lacZ mRNA secondary structures predicted with MFOLD. Altogether, these results indicate that mRNA binding to ribosome plays a major role in the enhancement of translation by adenine-rich codons irrespective of codon usage. This effect is naturally expressed in wild type systems and depends on adenine content, in contrast to the inhibition caused after over-expressing the lacZ derivatives containing rare codons in Pth defective cells. (C) 2011 Elsevier Masson SAS. All rights reserved.