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  • 1
    Language: English
    In: Nucleic acids research, 19 August 2016, Vol.44(14), pp.6907-23
    Description: Post-transcriptional regulation of transcription factors contributes to regulatory circuits. We created translational reporter fusions for multiple central regulators in Escherichia coli and examined the effect of Hfq-dependent non-coding RNAs on these fusions. This approach yields an 'RNA landscape,' identifying Hfq-dependent sRNAs that regulate a given fusion. No significant sRNA regulation of crp or fnr was detected. hns was regulated only by DsrA, as previously reported. Lrp and SoxS were both found to be regulated post-transcriptionally. Lrp, ' L: eucine-responsive R: egulatory P: rotein,' regulates genes involved in amino acid biosynthesis and catabolism and other cellular functions. sRNAs DsrA, MicF and GcvB each independently downregulate the lrp translational fusion, confirming previous reports for MicF and GcvB. MicF and DsrA interact with an overlapping site early in the lrp ORF, while GcvB acts upstream at two independent sites in the long lrp leader. Surprisingly, GcvB was found to be responsible for significant downregulation of lrp after oxidative stress; MicF also contributed. SoxS, an activator of genes used to combat oxidative stress, is negatively regulated by sRNA MgrR. This study demonstrates that while not all global regulators are subject to sRNA regulation, post-transcriptional control by sRNAs allows multiple environmental signals to affect synthesis of the transcriptional regulator.
    Keywords: Gene Expression Regulation, Bacterial ; Transcription, Genetic ; Escherichia Coli -- Genetics ; Escherichia Coli Proteins -- Genetics ; Leucine-Responsive Regulatory Protein -- Genetics ; RNA, Bacterial -- Metabolism ; Trans-Activators -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 2
    In: Nucleic Acids Research, 2016, Vol. 44(20), pp.9650-9666
    Description: Two-component systems (TCS) and small regulatory RNAs (sRNAs) are both widespread regulators of gene expression in bacteria. TCS are in most cases transcriptional regulators. A large class of sRNAs act as post-transcriptional regulators of gene expression that modulate the translation and/or stability of target-mRNAs. Many connections have been recently unraveled between these two types of regulators, resulting in mixed regulatory circuits with poorly characterized properties. This study focuses on the negative feedback circuit that exists between the EnvZ-OmpR TCS and the OmrA/B sRNAs. We have shown that OmpR directly activates transcription from the omrA and omrB promoters, allowing production of OmrA/B sRNAs that target multiple mRNAs, including the ompR-envZ mRNA. This control of ompR-envZ by the Omr sRNAs does not affect the amount of phosphorylated OmpR, i.e. the presumably active form of the regulator. Accordingly, expression of robust OmpR targets, such as the ompC or ompF porin genes, is not affected by OmrA/B. However, we find that several OmpR targets, including OmrA/B themselves, are sensitive to changing total OmpR levels. As a result, OmrA/B limit their own synthesis. These findings unravel an additional layer of control in the expression of some OmpR targets and suggest the existence of differential regulation within the OmpR regulon.
    Keywords: Chemistry ; Anatomy & Physiology;
    ISSN: 0305-1048
    E-ISSN: 1362-4962
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  • 3
    Language: English
    In: Nucleic Acids Research, 07/20/2016, p.gkw642
    ISSN: 0305-1048
    E-ISSN: 1362-4962
    Source: Oxford University Press (via CrossRef)
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  • 4
    Language: English
    In: Nucleic acids research, December 2008, Vol.36(21), pp.6781-94
    Description: Small RNAs are widespread regulators of gene expression in numerous organisms. This study describes the mode of action of two redundant Escherichia coli sRNAs, OmrA and OmrB, that downregulate the expression of multiple targets, most of which encode outer membrane proteins. Our results show that both sRNAs directly interact with at least two of these target mRNAs, ompT and cirA, in the vicinity of the translation initiation region, consistent with control of these targets being dependent on both Hfq and RNase E. Interestingly, these interactions depend on short stretches of complementarity and involve the conserved 5' end of OmrA/B. A mutation in this region abolishes control of all OmrA/B targets tested thus far, thereby highlighting the crucial role of the OmrA/B 5' end. This allowed us, by looking for mRNA sequences complementary to the OmrA/B 5' end, to identify ompR as an additional direct target of these two sRNAs. Since the OmpR transcriptional regulator activates expression of both omrA and omrB genes, this newly identified control should result in an autoregulatory loop limiting the amount of OmrA/B sRNAs.
    Keywords: Gene Expression Regulation, Bacterial ; Escherichia Coli -- Genetics ; RNA, Untranslated -- Chemistry
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 5
    Language: English
    In: Nucleic acids research, 13 April 2006, Vol.34(7), pp.e52
    Description: Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.
    Keywords: Fluorescent Antibody Technique, Indirect ; Gene Expression Profiling -- Methods ; Oligonucleotide Array Sequence Analysis -- Methods ; RNA, Untranslated -- Analysis
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 6
    Language: English
    In: Nucleic acids research, 2006, Vol.34(9), pp.2791-802
    Description: Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.
    Keywords: Software ; Escherichia Coli -- Genetics ; RNA, Bacterial -- Chemistry ; RNA, Untranslated -- Chemistry
    ISSN: 03051048
    E-ISSN: 1362-4962
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