相关实验视频

Updated: Jan 10, 2026

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Published on: October 8, 2018

对于基质识别的tRNA氨基碳烯基转移酶TapT的分子基础

Wei-Yan Wang¹, Heng-Rui Liang¹, Yu-Cong Wu¹

¹Key Laboratory of Glyco-drug Research of Zhejiang Province, School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China.

Nucleic acids research

|November 20, 2025

概括

TapT酶通过acp3U对转移RNA (tRNA) 进行修改. 研究人员确定了与SAM结合的TapT结构,揭示了其基质结合和潜在应用的催化机制.

科学领域:

生物化学生化学
分子生物学分子生物学
结构生物学结构生物学

背景情况:

3-(3-amino-3-carboxypropyl) uridine (acp3U) 是一种在细菌和真核生物中发现的关键tRNA修饰.
TapT是负责Escherichia coli tRNA中acp3U修饰的酶,使用S-adenosylmethionine (SAM).
目前尚不清楚TapT的精确基质结合和催化机制.

研究的目的:

为了阐明TapT酶在SAM结合状态中的三维结构.
调查TapT的辅因子识别机制.
探索TapT.的tRNA识别机制.

主要方法:

进行X射线晶体学以确定TapT-SAM复合体的3D结构.
异热定位热量计 (ITC) 用于评估结合亲缘关系.
液体染色学-质谱学 (LC-MS) 用于酶活性检测.
光偏振测定用于tRNA识别研究.

主要成果:

确定的结构显示TapT采用SPOUT折叠,SAM绑在一个可适应的口袋中.
用S-甲基-5'-thioadenosine和 sinefungin进一步表征了辅因子的识别.
测量了结合亲和和和酶活性,并探索了tRNA识别机制.

结论:

该研究为TapT基质识别及其催化机制提供了详细的分子基础.
了解TapT的功能可以促进生物化学,分子生物学和潜在的瘤诊断方面的研究.

更多相关视频

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

Published on: June 21, 2017

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

Published on: December 29, 2021

相关概念视频

Transfer RNA Synthesis

Transfer RNA Synthesis

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...

Transfer RNA Synthesis

Transfer RNA Synthesis

tRNA Activation

tRNA Activation

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...

tRNA Activation

tRNA Activation

Improving Translational Accuracy

Improving Translational Accuracy

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...

Allosteric Proteins-ATCase

Allosteric Proteins-ATCase

Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...