PL EN


Preferences help
enabled [disable] Abstract
Number of results
2018 | 65 | 1 | 141-149
Article title

A search for the in trans role of GraL, an Escherichia coli small RNA

Content
Title variants
Languages of publication
EN
Abstracts
EN
Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL's targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusion's β-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusion's activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL's mechanism of action is non-canonical, unique and requires further research.
Keywords
Publisher

Year
Volume
65
Issue
1
Pages
141-149
Physical description
Dates
published
2018
received
2018-02-01
revised
2018-02-26
accepted
2018-03-03
(unknown)
2018-03-12
Contributors
  • Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdansk, Gdańsk, Poland
  • Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdansk, Gdańsk, Poland
  • Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdansk, Gdańsk, Poland
References
  • Aberg A, Shingler V, Balsalobre C (2008) Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo. Mol Microbiol 67: 1223-1241. doi: 10.1111/j.1365-2958.2006.05191.x.
  • Beisel CL, Updegrove TB, Janson BJ, Storz G (2012) Multiple factors dictate target selection by Hfq-binding small RNAs. EMBO J 31: 1961-1974. doi: 10.1038/emboj.2012.52.
  • Bernhardt TG, de Boer PA (2005) SlmA, a nucleoid-associated, FtsZ binding protein required for blocking septal ring assembly over chromosomes in E. coli. Mol Cell 18: 555-564. doi: 10.1016/j.molcel.2005.04.012.
  • Borukhov S, Lee J, Laptenko O (2005) Bacterial transcription elongation factors: new insights into molecular mechanism of action. Mol Microbiol 55: 1315-1324. doi: 10.1111/j.1365-2958.2004.04481.x.
  • Churchward G, Belin D, Nagamine Y (1984) A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene 31: 165-171. doi: 10.1016/0378-1119(84)90207-5.
  • de Boer PA, Crossley RE, Rothfield LI (1989) A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell 56: 641-649. doi: 10.1016/0092-8674(89)90586-2.
  • Ducey TF, Dyer DW (2002) Rapid identification of EZ::TN™ transposon insertion sites in the genome of Neisseria gonorrhoeae. EPICENTRE Forum 9: 6-7.
  • Faner MA, Feig AL (2013) Identifying and characterizing Hfq-RNA interactions. Methods 63: 144-159. doi: 10.1016/j.ymeth.2013.04.023.
  • Fender A, Elf J, Hampel K, Zimmermann B, Wagner EG (2010) RNAs actively cycle on the Sm-like protein Hfq. Genes Dev 24: 2621-2626. doi: 10.1101/gad.591310.
  • Foster JW, Park YK, Penfound T, Fenger T, Spector MP (1990) Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon. J Bacteriol 172: 4187-4196.
  • Gottesman S (2004) The small RNA regulators of Escherichia coli: roles and mechanisms. Annu Rev Microbiol 58: 303-328. doi: 10.1146/annurev.micro.58.030603.123841.
  • Gottesman S, Storz G (2011) Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harb Perspect Biol 3: a003798. doi: 10.1101/cshperspect.a003798.
  • Guillier M, Gottesman S (2006) Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs. Mol Microbiol 59: 231-247. doi: 10.1111/j.1365-2958.2005.04929.x.
  • Hoe CH, Raabe CA, Rozhdestvensky TS, Tang TH (2013) Bacterial sRNAs: Regulation in stress. Int J Med Microbiol 303: 217-229. doi: 10.1016/j.ijmm.2013.04.002.
  • Holder IT, Wagner S, Xiong P, Sinn M, Frickey T, Meyer A, Hartig JS (2015) Intrastrand triplex DNA repeats in bacteria: a source of genomic instability. Nucleic Acids Res 43: 10126-10142. doi: 10.1093/nar/gkv1017.
  • Huang HY, Chang HY, Chou CH, Tseng CP, Ho SY, Yang CD, Ju YW, Huang HD (2009) sRNAMap: genomic maps for small non-coding RNAs, their regulators and their targets in microbial genomes. Nucleic Acids Res 37: D150-D154. doi: 10.1093/nar/gkn852.
  • Kawano M, Oshima T, Kasai H, Mori H (2002) Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli. Mol Microbiol 45: 333-349. doi: 10.1046/j.1365-2958.2002.03042.x.
  • Kery MB, Feldman M, Livny J, Tjaden B (2014) TargetRNA2: identifying targets of small regulatory RNAs in bacteria. Nucleic Acids Res 42: W124-W129. doi: 10.1093/nar/gku317.
  • Kortmann J, Narberhaus F. (2012) Bacterial RNA thermometers: molecular zippers and switches. Nat Rev Microbiol 10: 255-265. doi: 10.1038/nrmicro2730.
  • Laptenko O, Lee J, Lomakin I, Borukhov S (2003) Transcript cleavage factors GreA and GreB act as a transient catalytic components of RNA polymerase. EMBO J 22: 6322-6334. doi: 10.1093/emboj/cdg610.
  • Lease RA, Cusick ME, Belfort M (1998) Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. Proc Natl Acad Sci U S A 95: 12456-12461. doi: 10.1073/pnas.95.21.12456.
  • Majdalani N, Vanderpool CK, Gottesman S (2005) Bacterial small RNA regulators. Crit Rev Biochem Mol Biol 40: 93-113. doi: 10.1080/10409230590918702.
  • Massé E, Salvail H, Desnoyers G, Arguin M (2007) Small RNAs controlling iron metabolism. Curr Opin Microbiol 10: 140-145. doi: 10.1016/j.mib.2007.03.013.
  • Masse E, Vanderpool CK, Gottesman S (2005) Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187: 6962-6971. doi: 10.1128/JB.187.20.6962-6971.2005.
  • McCullen CA, Benhammou JN, Majdalani N, Gottesman S (2010) Mechanism of positive regulation by DsrA and RprA small noncoding RNAs: pairing increases translation and protects rpoS mRNA from degradation. J Bacteriol 192: 5559-5571. doi: 10.1128/JB.00464-10.
  • Melamed S, Peer A, Faigenbaum-Romm R, Gatt YE, Reiss N, Bar A, Altuvia Y, Argaman L, Margalit H (2016) Global mapping of small RNA-target interactions in bacteria. Mol Cell 63: 884-897. doi: 10.1016/j.molcel.2016.07.026.
  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • Morita T, Maki K, Aiba H (2012) Detection of sRNA-mRNA interactions by electrophoretic mobility shift assay. Methods Mol Biol 905: 235-244. doi: 10.1007/978-1-61779-949-5_15.
  • Moon K, Gottesman S (2009) A PhoQ/P-regulated small RNA regulates sensitivity of Escherichia coli to antimicrobial peptides. Mol Microbiol 74: 1314-1330. doi: 10.1111/j.1365-2958.2009.06944.x.
  • Moon K, Gottesman S (2011) Competition among Hfq-binding small RNAs in Escherichia coli. Mol Microbiol 82: 1545-1562. doi: 10.1111/j.1365-2958.2011.07907.x.
  • O'Handley SF, Frick DN, Dunn CA, Bessman MJ (1998) Orf186 represents a new member of the Nudix hydrolases, active on adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH. J Biol Chem 273: 3192-3197.
  • Pedersen K, Gerdes K (1999) Multiple hok genes on the chromosome of Escherichia coli. Mol Microbiol 32: 1090-1102. doi: 10.1046/j.1365-2958.1999.01431.x.
  • Potrykus K, Cashel M (2008) (p)ppGpp: still magical? Annu Rev Microbiol 62: 35-51. doi: 10.1146/annurev.micro.62.081307.162903.
  • Potrykus K, Murphy H, Chen X, Epstein JA, Cashel M (2010) Imprecise transcription termination within Escherichia coli greA leader gives rise to an array of short transcripts, GraL. Nucleic Acids Res 38: 1636-1651. doi: 10.1093/nar/gkp1150.
  • Potrykus K, Vinella D, Murphy H, Szalewska-Palasz A, D'Ari R, Cashel M (2006) Antagonistic regulation of Escherichia coli ribosomal RNA rrnB P1 promoter activity by GreA and DksA. J Biol Chem 281: 15238-15248. doi: 10.1074/jbc.M601531200.
  • Powell BS, Rivas MP, Court DL, Nakamura Y, Turnbough CL Jr. (1994) Rapid confirmation of single copy lambda prophage integration by PCR. Nucleic Acids Res 22: 5765-5766.
  • Romby P, Vandenesch F, Wagner EG (2006) The role of RNAs in the regulation of virulence-gene expression. Curr Opin Microbiol 9: 229-236. doi: 10.1016/j.mib.2006.02.005.
  • Sedlyarova N, Rescheneder P, Magan A, Popitsch N, Rziha N, Bilusic I, Epshtein V, Zimmermann B, Lybecker M, Sedlyarov V, Schroeder R, Nudler E (2017) Natural RNA polymerase aptamers regulate transcription in E. coli. Mol Cell 67: 30-43. doi: 10.1016/j.molcel.2017.05.025.
  • Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4: 206-223. doi: 10.1038/nprot.2008.227.
  • Simons RW, Houman F, Kleckner N (1987) Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53: 85-96. doi: 10.1016/0378-1119(87)90095-3.
  • Sledjeski D, Gottesman S (1995) A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli. Proc Natl Acad Sci U S A 92: 2003-2007.
  • Smirnov A, Forstner KU, Holmqvist E, Otto A, Gunster R, Becher D, Reinhardt R, Vogel J (2016) Grad-seq guides the discovery of ProQ as a major small RNA-binding protein. Proc Natl Acad Sci U S A 113: 11591-11596. doi: 10.1073/pnas.1609981113.
  • Storz G, Vogel J, Wassarman KM (2011) Regulation by Small RNAs in Bacteria: Expanding Frontiers. Mol Cell 43: 880-891. doi: 10.1016/j.molcel.2011.08.022.
  • Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219: 37-44. doi: 10.1016/0022-2836(91)90855-Z.
  • Tjaden B, Goodwin SS, Opdyke JA, Guillier M, Fu DX, Gottesman S, Storz G (2006) Target prediction for small, noncoding RNAs in bacteria. Nucleic Acids Res 34: 2791-2802. doi: 10.1093/nar/gkl356.
  • Toledo-Arana A, Repoila F, Cossart P (2007) Small noncoding RNAs controlling pathogenesis. Curr Opin Microbiol 10: 182-188. doi: 10.1016/j.mib.2007.03.004.
  • Tu K, Bassler BL (2007) Multiple small RNAs act additively to integrate sensory information and control quorum sensing in Vibrio harveyi. Genes Dev 21: 221-233. doi: 10.1101/gad.1502407.
  • Valentin-Hansen P, Johansen J, Rasmussen AA (2007) Small RNAs controlling outer membrane porins. Curr Opin Microbiol 10: 152-155. doi: 10.1016/j.mib.2007.03.001.
  • Vecerek B, Moll I, Bläsi U (2007) Control of Fur synthesis by the non-coding RNA RyhB and iron-responsive decoding. EMBO J 26: 965-975. doi: 10.1038/sj.emboj.7601553.
  • Vinella D, Potrykus K, Murphy H, Cashel M (2012) Effects on growth by changes of the balance between GreA, GreB, and DksA suggest mutual competition and functional redundancy in Escherichia coli. J Bacteriol 194: 261-273. doi: 10.1128/JB.06238-11.
  • Wright PR, Georg J, Mann M, Sorescu DA, Richter AS, Lott S, Kleinkauf R, Hess WR, Backofen R (2014) CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res 42: W119-W123. doi: 10.1093/nar/gku359.
  • Ying X, Cao Y, Wu J, Liu Q, Cha L, Li W (2011) sTarPicker: a method for efficient prediction of bacterial sRNA targets based on a two-step model for hybridization. PLoS One 6: e22705. doi: 10.1371/journal.pone.0022705.
  • Zhou J, Rudd K (2013) EcoGene 3.0. Nucleic Acids Res 41: D613-D624. doi: 10.1093/nar/gks1235.
Document Type
Publication order reference
Identifiers
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv65p141kz
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.