Transmission line model analysis of transcription factors binding to oligoduplexes – differentiation of the effect of single nucleotide modifications

Rodrigues, Rogério M. M.; de-Carvalho, Jorge; Henriques, Sílvia F.; Mira, Nuno P.; Sá‐Correia, Isabel; Ferreira, Guilherme N. M.

doi:10.1039/c4an00709c

Cited by 2 publications

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“…The Haa1/HRE recognition process was examined using a new Quartz Crystal Microbalance (QCM) analytical method and a transmission line model (TLM) algorithm to characterize the mechanical properties of the Haa1/DNA complex and the effect of single point mutations on this recognition process [20, 21]. Among all genes upregulated by acetic acid exposure that are dependent on Haa1 expression, 55% contain the HRE motif in the promoter region, and are considered to be direct targets of Haa1.…”

Section: Introductionmentioning

confidence: 99%

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms

et al. 2017

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BackgroundBesides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the transcription factor Haa1 is an important determinant of the tolerance to this acid. The engineering of Haa1 either by overexpression or mutagenesis has therefore been considered to be a promising avenue towards the construction of more robust strains with improved acetic acid tolerance.ResultsBy applying the concept of global transcription machinery engineering to the regulon-specific transcription factor Haa1, a mutant allele containing two point mutations could be selected that resulted in a significantly higher acetic acid tolerance as compared to the wild-type allele. The level of improvement obtained was comparable to the level obtained by overexpression of HAA1, which was achieved by introduction of a second copy of the native HAA1 gene. Dissection of the contribution of the two point mutations to the phenotype showed that the major improvement was caused by an amino acid exchange at position 135 (serine to phenylalanine). In order to further study the mechanisms underlying the tolerance phenotype, Haa1 translocation and transcriptional activation of Haa1 target genes was compared between Haa1 mutant, overproduction and wild-type strains. While the rapid Haa1 translocation from the cytosol to the nucleus in response to acetic acid was not affected in the Haa1S135F mutant strain, the levels of transcriptional activation of four selected Haa1-target genes by acetic acid were significantly higher in cells of the mutant strain as compared to cells of the wild-type strain. Interestingly, the time-course of transcriptional activation in response to acetic acid was comparable for the mutant and wild-type strain whereas the maximum mRNA levels obtained correlate with each strain’s tolerance level.ConclusionOur data confirms that engineering of the regulon-specific transcription factor Haa1 allows the improvement of acetic acid tolerance in S. cerevisiae. It was also shown that the beneficial S135F mutation identified in the current work did not lead to an increase of HAA1 transcript level, suggesting that an altered protein structure of the Haa1S135F mutant protein led to an increased recruitment of the transcription machinery to Haa1 target genes.

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confidence: 99%

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms

et al. 2017

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“…In recent years, emerging assays for TFs detection include fluorescent, − colorimetric, − and electrochemical methods. − Among all these methods, fluorescent assays are attracting accumulating attention due to high sensitivity. Heyduk’s group pioneered fluorescent detection of TFs using two DNA duplexes each containing half of a TFs binding sequence .…”

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Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification

Chen

et al. 2017

Anal. Chem.

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Transcription factors (TFs) regulate gene expression by binding to regulatory regions, and their dysregulation is involved in numerous diseases. Thus, they are therapeutic targets and potential diagnostic markers. However, widely used methods for TFs detection are either cumbersome or costly. Herein, we first applied DNA-Ag nanoclusters molecular beacons (AgMBs) in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs. In the absence of TFs, a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe (referred as dsTFs probe) under exonuclease III (Exo III) digestion. Then, the reporter triggers downstream Exo III-assisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs, resulting in significant fluorescent decrease eventually. Conversely, the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state. To testify this rationale, we utilized nuclear factor-kappa B p50 (NF-κB p50) as a model TFs. Owing to the amplification strategy, this method exhibited high sensitivity toward NF-κB p50 with a limit of detection of 10 pM, and a broad linear range from 30 pM to 1.5 nM. Furthermore, this method could detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation. Finally, by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC of corresponding inhibitors.

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Transmission line model analysis of transcription factors binding to oligoduplexes – differentiation of the effect of single nucleotide modifications

Cited by 2 publications

References 22 publications

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms

Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification

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