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相关概念视频

Improving Translational Accuracy02:07

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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...
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Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
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Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
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Lesson: Translation
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
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克服人类细胞衍生翻译系统中的eIF2α制动

Nikolay A Aleksashin1,2, Rohan R Shelke2,3, Tianhao Yin2,4

  • 1Innovative Genomics Institute, University of California, Berkeley, CA, USA.

bioRxiv : the preprint server for biology
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PubMed
概括
此摘要是机器生成的。

欧核细胞启动因子2α (eIF2α) 的抑制酸化限制了人类细胞无转化. 诸如基因组编辑或GADD34/K3L表达等策略克服了这一障碍,增强了合成生物学应用.

关键词:
在Expi293F中使用.在GADD34中,GADD34是什么意思?K3LL 在线播放这就是PKR和PKR.这就是WI-38的原因.这是心肌细胞 (cardiomyocyte).没有细胞的蛋白质合成.eIF2α的酸化方式这就是 iPSC 的意义.在体外翻译 in vitro翻译控制翻译启动的控制

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科学领域:

  • 分子生物学分子生物学
  • 细胞生物学 细胞生物学
  • 合成生物学 合成生物学

背景情况:

  • 来自人类细胞的无细胞翻译系统对于研究基因表达和开发合成生物学工具至关重要.
  • 这些系统的生产率通常受到Ser52残留物中真核细胞启动因子2α (eIF2α) 的抑制酸化的限制.

研究的目的:

  • 在可编辑和难以编辑的人类细胞类型中系统地探索和比较绕过eIF2α酸化媒介启动块的策略.
  • 确定产生高活性人体细胞无转化提取物的最佳方法.

主要方法:

  • 对EIF2S1进行基因组编辑,在Expi293F细胞中产生eIF2α-S52A突变.
  • 在Expi293F细胞中EIF2AK2 (PKR) 的遗传淘汰.
  • 在iPSC和初级纤维细胞中,在Tet诱导性控制下,稳定 piggyBac 整合截断的 GADD34 (PPP1R15A) 和 K3L.
  • 工程化iPSCs分化为心肌细胞,用于提取物生产.

主要成果:

  • 在Expi293F细胞中进行基因组编辑以阻止eIF2α Ser52酸化 (eIF2α-S52A),显著增加了翻译提取物的活性.
  • EIF2AK2 (PKR) 的淘汰也增强了Expi293F溶酸盐的翻译,证实了eIF2α酸化是关键的瓶.
  • 在iPSC (包括心肌细胞) 和初级纤维细胞中通过piggyBac系统表达GADD34和K3L成功改善了翻译输出.
  • 这些基于表达的方法为基因组编辑无法实现的系统提供了可行的替代方案.

结论:

  • eIF2α酸化是人类无细胞提取物中强大的翻译的主要障碍.
  • 编辑eIF2α或PKR淘汰的基因组编辑是可编辑细胞系统的最佳选择.
  • 一个便携式的GADD34/K3L表达盒可从具有挑战性或不可编辑的系统中产生翻译活性溶解体,从而扩大了无人细胞翻译的实用性.