NTPs compete in the active site of RNA polymerases I and II

IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Biophysical chemistry Pub Date : 2024-08-03 DOI:10.1016/j.bpc.2024.107302
Kaila B. Fuller , Ryan M. Requijo , David A. Schneider , Aaron L. Lucius
{"title":"NTPs compete in the active site of RNA polymerases I and II","authors":"Kaila B. Fuller ,&nbsp;Ryan M. Requijo ,&nbsp;David A. Schneider ,&nbsp;Aaron L. Lucius","doi":"10.1016/j.bpc.2024.107302","DOIUrl":null,"url":null,"abstract":"<div><p>Eukaryotes express at least three RNA polymerases (Pols) carry out transcription, while bacteria and archaea use only one. Using transient state kinetics, we have extensively examined and compared the kinetics of both single and multi-nucleotide additions catalyzed by the three Pols. In single nucleotide addition experiments we have observed unexpected extension products beyond one incorporation, which can be attributed to misincorporation, the presence of nearly undetectable amounts of contaminating NTPs, or a mixture of the two. Here we report the development and validation of an analysis strategy to account for the presence of unexpected extension products, when they occur. Using this approach, we uncovered evidence showing that non-cognate nucleotide, thermodynamically, competes with cognate nucleotide for the active site within the elongation complex of Pol I, ΔA12 Pol I, and Pol II. This observation is unexpected because base pairing interactions provide favorable energetics for selectivity and competitive binding indicates that the affinities of cognate and non-cognate nucleotides are within an order of magnitude. Thus, we show that application of our approach will allow for the extraction of additional information that reports on the energetics of nucleotide entry and selectivity.</p></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"314 ","pages":"Article 107302"},"PeriodicalIF":3.3000,"publicationDate":"2024-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biophysical chemistry","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0301462224001315","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Eukaryotes express at least three RNA polymerases (Pols) carry out transcription, while bacteria and archaea use only one. Using transient state kinetics, we have extensively examined and compared the kinetics of both single and multi-nucleotide additions catalyzed by the three Pols. In single nucleotide addition experiments we have observed unexpected extension products beyond one incorporation, which can be attributed to misincorporation, the presence of nearly undetectable amounts of contaminating NTPs, or a mixture of the two. Here we report the development and validation of an analysis strategy to account for the presence of unexpected extension products, when they occur. Using this approach, we uncovered evidence showing that non-cognate nucleotide, thermodynamically, competes with cognate nucleotide for the active site within the elongation complex of Pol I, ΔA12 Pol I, and Pol II. This observation is unexpected because base pairing interactions provide favorable energetics for selectivity and competitive binding indicates that the affinities of cognate and non-cognate nucleotides are within an order of magnitude. Thus, we show that application of our approach will allow for the extraction of additional information that reports on the energetics of nucleotide entry and selectivity.

Abstract Image

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
NTP 在 RNA 聚合酶 I 和 II 的活性位点上相互竞争
真核生物至少表达三种 RNA 聚合酶(Pols)进行转录,而细菌和古细菌只使用一种。利用瞬态动力学,我们对三种 Pols 催化的单核苷酸和多核苷酸添加的动力学进行了广泛的研究和比较。在单核苷酸加成实验中,我们观察到了超出一次加成的意外延伸产物,这可能是由于误加成、存在几乎检测不到的污染 NTP 或两者的混合所致。在此,我们报告了一种分析策略的开发和验证情况,该策略可在出现意外延伸产物时对其进行解释。利用这种方法,我们发现有证据表明,在 Pol I、ΔA12 Pol I 和 Pol II 的延伸复合体中,非识别核苷酸在热力学上与识别核苷酸竞争活性位点。这一观察结果出乎意料,因为碱基配对相互作用为选择性提供了有利的能量,而竞争性结合表明同源核苷酸和非同源核苷酸的亲和力在一个数量级之内。因此,我们的研究表明,应用我们的方法可以提取更多信息,报告核苷酸进入和选择性的能量学。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
Biophysical chemistry
Biophysical chemistry 生物-生化与分子生物学
CiteScore
6.10
自引率
10.50%
发文量
121
审稿时长
20 days
期刊介绍: Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.
期刊最新文献
Functional characterization of Staphylococcus aureus lipase 2 (SAL2) as a collagen adhesin. Urineprint of high-altitude: Insights from analyses of urinary biomarkers and bio-physical-chemical features of extracellular vesicles Kinetics of i-motif folding within the duplex context Supramolecular arrangements in human amyloid tissues using SAXS Characterization of a novel salt- and solvent-tolerant esterase Dhs82 from soil metagenome capable of hydrolyzing estrogenic phthalate esters
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1