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Nucleic Acid Measurements Video

DNA Quantitation Using a Spectrophotometer

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The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. YoeB—YefM, the widespread type II toxin—antitoxin TA module, binds to its own Nucleic Acid Measurements to autoregulate its transcription: Measuremenfs or induce transcription under normal or stress conditions, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain.

INTRODUCTION

In contrast, at TA ratio ofbinding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which Measugements unable to bind the promoter DNA On Open Market New A Paradigm The due to steric clashes, hence derepresses TA operon transcription. Toxin—antitoxin TA systems, the widespread genetic modules in bacterial genomes and plasmids, are emerging as key players in stress responses 1—4. TA gene loci were originally found on plasmids that provided growth advantage for bacteria with TA-containing plasmids and killed plasmid-free bacteria via a mechanism called post-segregation killing PSK 56. Since then, TA operons Meaeurements been found not only in plasmids, but also on the chromosomes of most free-living bacteria to facilitate bacteria cell survival under various stress conditions 7.

TA system consists of a gene pair encoding a stable toxin that impedes the cell growth by interfering with the vital cellular processes such as transcription, translation, DNA replication and membrane homeostasis, and an unstable antitoxin that counteracts the toxin activity 89. Currently, six classes of TA system have been reported on the basis of mechanism for neutralizing the biological effect of toxins Nucleic Acid Measurements Type II TA system is the most widely studied module found in most of the free-living bacteria 12— Most of the type II toxins are endoribonucleases that adopt microbial RNase fold Antitoxins are regulatory proteins that are typically Nucleic Acid Measurements of two domains, i.

N-terminal DNA binding domain and Nucleic Acid Measurements toxin neutralizing domain that is intrinsically disordered 10 Some of type II TA modules are subject to transcriptional autoregulation to ensure that toxins are repressed transcriptionally under normal growth condition, and are induced only when cells are under stress 1619 Nucleic Acid Measurements core concept of conditional cooperativity is that the level of TA operon transcription is controlled by the ratio of toxin to antitoxin, and toxin acts as a co-repressor or de-repressor for the antitoxin at lower or higher molar ratio to either suppress or derepress transcription 1021— Previous research demonstrated that the YoeB—YefM complex plays a key role in processes such as biofilm formation and response to oxidative stress YoeB—YefM from Escherichia Acud was first identified as a homolog of axe-txe from Nuclejc faecium In addition, the structure of YoeB in complex with 30S and 70S ribosomes shed light to its mechanism of mRNA cleaving recently 24 However, the underlying structural mechanism remains elusive: i.

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In the present work, we find S. Interestingly, the structures clearly show YoeB toxin binding induced the intrinsic disordered to ordered transition of YefM C-terminal domain. Moreover, structural analysis explained why the heterohexamer state can optimally bind to the promoter DNA whereas the heterotetramer cannot. Our work provides detail molecular insights into understanding Measureemnts complex mediated transcriptional autoregulation, which will help target this type II TA system to overcome antibiotics resistance in S.

Nucleic Acid Measurements plasmids were derived from the corresponding plasmids via site-directed mutagenesis.

All plasmids used in this study are listed in the Supplementary Table S3. The plasmid containing the full-length yefM was transformed into E.

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Different combination of plasmids were co-transformed into E. The same strategy was also applied for the expression of site-mutation heterohexameric complex.

Due to the YoeB cytotoxicity for E. The refolded YoeB protein was further purified using size-exclusion chromatography.

Carbohydrates, Lipids, Proteins, And Nucleic Acids

Molecular Nucleic Acid Measurements method was used to determine the initial phase with Phenix. The segment YoeB and YefM residues of 40—83 of heterotetramer was used as search model to solve the structure of heterohexamer-DNA, and the model was further completed after several iterations of automatic and manual building with Phenix. The YoeB monomer from heterotetramer was used to solve the structure of YoeB-dimer. Refinement was performed by combining Phenix. The data collection and structure refinement statistics are summarized in Supplementary Table S1. For each measurement, 20 Nucleic Acid Measurements frames of 1 s exposure time were recorded. Similarly, the background data were recorded in the same buffer and were subtracted from the protein patterns.]