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      Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts

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          Abstract

          Regulatory RNA regions within a transcript, particularly in the 5′ untranslated region (5′UTR), have been shown in a variety of organisms to control the expression levels of these mRNAs in response to various metabolites or environmental conditions. Considering the unique tolerance of Zymomonas mobilis to ethanol and the growing interest in engineering microbial strains with enhanced tolerance to industrial inhibitors, we searched natural cis-regulatory regions in this microorganism using transcriptomic data and bioinformatics analysis. Potential regulatory 5′UTRs were identified and filtered based on length, gene function, relative gene counts, and conservation in other organisms. An in vivo fluorescence-based screening system was developed to confirm the responsiveness of 36 5′UTR candidates to ethanol, acetate, and xylose stresses. UTR_ZMO0347 (5′UTR of gene ZMO0347 encoding the RNA binding protein Hfq) was found to down-regulate downstream gene expression under ethanol stress. Genomic deletion of UTR_ZMO0347 led to a general decrease of hfq expression at the transcript level and increased sensitivity for observed changes in Hfq expression at the protein level. The role of UTR_ZMO0347 and other 5′UTRs gives us insight into the regulatory network of Z. mobilis in response to stress and unlocks new strategies for engineering robust industrial strains as well as for harvesting novel responsive regulatory biological parts for controllable gene expression platforms in this organism.

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          Most cited references43

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          Rfam: an RNA family database.

          Rfam is a collection of multiple sequence alignments and covariance models representing non-coding RNA families. Rfam is available on the web in the UK at http://www.sanger.ac.uk/Software/Rfam/ and in the US at http://rfam.wustl.edu/. These websites allow the user to search a query sequence against a library of covariance models, and view multiple sequence alignments and family annotation. The database can also be downloaded in flatfile form and searched locally using the INFERNAL package (http://infernal.wustl.edu/). The first release of Rfam (1.0) contains 25 families, which annotate over 50 000 non-coding RNA genes in the taxonomic divisions of the EMBL nucleotide database.
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            Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression.

            Although proteins fulfil most of the requirements that biology has for structural and functional components such as enzymes and receptors, RNA can also serve in these capacities. For example, RNA has sufficient structural plasticity to form ribozyme and receptor elements that exhibit considerable enzymatic power and binding specificity. Moreover, these activities can be combined to create allosteric ribozymes that are modulated by effector molecules. It has also been proposed that certain messenger RNAs might use allosteric mechanisms to mediate regulatory responses depending on specific metabolites. We report here that mRNAs encoding enzymes involved in thiamine (vitamin B(1)) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the need for protein cofactors. The mRNA-effector complex adopts a distinct structure that sequesters the ribosome-binding site and leads to a reduction in gene expression. This metabolite-sensing regulatory system provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.
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              Gene regulation by riboswitches.

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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                08 December 2017
                2017
                : 8
                : 2432
                Affiliations
                [1] 1Institute for Cellular and Molecular Biology, University of Texas at Austin , Austin, TX, United States
                [2] 2Department of Chemical Engineering, Cockrell School of Engineering, University of Texas at Austin , Austin, TX, United States
                [3] 3Hubei Key Laboratory of Industrial Biotechnology, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University , Wuhan, China
                Author notes

                Edited by: Xueyang Feng, Virginia Tech, United States

                Reviewed by: Fei Tao, Shanghai Jiao Tong University, China; Aindrila Mukhopadhyay, Lawrence Berkeley National Laboratory, United States

                *Correspondence: Lydia M. Contreras lcontrer@ 123456che.utexas.edu

                This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.02432
                5770649
                a1a07136-3722-48ca-a6a4-b44d07787241
                Copyright © 2017 Cho, Haning, Shen, Blome, Li, Yang and Contreras.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 26 July 2017
                : 23 November 2017
                Page count
                Figures: 6, Tables: 1, Equations: 0, References: 57, Pages: 15, Words: 9385
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                regulatory rna,5′ utr,zymomonas mobilis,5′ rapid amplification of cdna ends (race),stress response

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