用于哺乳动物蛋白质合成的细胞选择性分析的工程氨基基-tRNA合成酶
Alborz Mahdavi, Graham D Hamblin, Granton A Jindal
1Max Planck Institute for Brain Research , Frankfurt am Main 60438, Germany.
Journal of the American Chemical Society
|March 19, 2016
概括
研究人员开发了一种新的工具,即突变的甲基-tRNA合成酶 (L274GMmMetRS),用于细胞选择性蛋白质标记. 这种方法可以在没有复杂程序的情况下精确分析哺乳动物细胞中的蛋白质合成.
科学领域:
- 生物化学
- 分子生物学
- 细胞生物学
背景情况:
- 在多细胞生物体中研究蛋白质组动力学需要细胞选择性分析方法.
- 现有的蛋白质合成分析方法可能复杂且劳动密集.
研究的目的:
- 开发一种用于细胞选择性标记和分析哺乳动物细胞中新合成的蛋白质的新工具.
- 为了证明突变的甲基-tRNA合成酶在将非正规氨基酸纳入蛋白质中的实用性.
主要方法:
- 一种突变的小鼠甲基-tRNA合成酶 (L274GMmMetRS) 已被设计成将阿齐多诺素 (Anl) 充电到延长 tRNA ((Met)).
- 这种工程酶在子 (CHO),子 (COS7) 和人类 (HeLa) 细胞系中得到表达.
- 用Anl标记表达L274GMmMetRS的细胞,并通过质谱测量标记和识别所产生的蛋白质.
主要成果:
- 在多种哺乳动物细胞系 (CHO,COS7,HeLa) 中,L274GMmMetRS成功充电了阿齐多诺素 (Anl).
- 在突变合成酶的存在下合成的蛋白质可以被选择性标记为Anl.
- 该方法使用质谱学促进了蛋白质丰富和鉴定,而不需要正交tRNA或氨基酸耗尽.
结论:
- 在哺乳动物系统中,L274GMmMetRS酶是细胞选择性蛋白质标记的有效工具.
- 这种方法简化了细胞层面的蛋白质合成动态分析.
- 这种方法为在复杂的生物环境中研究蛋白质组动态提供了一个多功能平台.
相关概念视频
02:26tRNA Activation
23.8K
Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
23.8K
02:26tRNA Activation
8.9K
8.9K
02:07Improving Translational Accuracy
15.4K
Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
15.4K
02:36Transfer RNA Synthesis
13.7K
One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
13.7K
02:24Ribosome Profiling
4.3K
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
4.3K