Skip to main content
SpringerLink
Log in

The 5′ untranslated region of the soybean cytosolic glutamine synthetase β1 gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In plants, it occurs as two major isoforms, a cytosolic form (GS1) and a nuclear encoded chloroplastic form. The focus of this paper is to determine the role of the 5′UTR of a GS1 gene. GS1 gene constructs with and without its 5′ and 3′ UTRs, driven by a constitutive promoter, were agroinfiltrated into tobacco leaves and the tissues were analyzed for both transgene transcript and protein accumulation. The constructs were also tested in an in vitro transcription/translation system and in Escherichia coli. Our results showed that while the 3′UTR functioned in the destabilization of the transcript, the 5′UTR acted as a translation enhancer in plant cells but not in the in vitro translation system. The 5′UTR of the GS1 gene when placed in front of a reporter gene (uidA), showed a 20-fold increase in the level of GUS expression in agroinfiltrated leaves when compared to the same gene construct without the 5′UTR. The 5′UTR-mediated translational enhancement is probably another step in the regulation of GS in plants. The presence of the GS1 5′UTR in front of the GS1 coding region allowed for its translation in E. coli suggesting the commonality of the translation initiation mechanism for this gene between plants and bacteria.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Agalarov SC, Sogorin EA, Shirokikh NE, Spirin AS (2011) Insight into the structural organization of the omega leader of TMV RNA: the role of various regions of the sequence in the formation of a compact structure of the omega RNA. Biochem Biophys Res Commun 404:250–253

    Article  PubMed  CAS  Google Scholar 

  • Beltran-Peña E, Aguilar R, Ortíz-Lopez A, Dinkova TD, Sánchez de Jiménez E (2002) Auxin stimulates S6 ribosomal protein phosphorylation in maize thereby affecting protein synthesis regulation. Physiol Plant 115:291–297

    Article  PubMed  Google Scholar 

  • Bennett M, Cullimore J (1990) Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes, and complementation of a glnA mutant. Eur J Biochem 193:319–324

    Article  PubMed  CAS  Google Scholar 

  • Bernard SM, Habash DZ (2009) The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol 182:608–620

    Article  PubMed  CAS  Google Scholar 

  • Carvalho H, Sunkel C, Salema R, Cullimore JV (1997) Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA mutant with a plant domain-swapped enzyme. Plant Mol Biol 35:623–632

    Article  PubMed  CAS  Google Scholar 

  • Dansako T, Kato K, Satoh J, Sekine M, Yoshida K, Shinmyo A (2003) 5′untranslated region of the HSP18.2 gene contributes to efficient translation in plant cells. J Biosci Bioeng 95:52–58

    PubMed  CAS  Google Scholar 

  • DasSarma S, Tisher E, Goodman HM (1986) Plant glutamine synthetase complements a glnA mutation in Escherichia coli. Science 232:1242–1244

    Article  PubMed  CAS  Google Scholar 

  • Diaz C, Kusano M, Sulpice R, Araki M, Redestig H, Saito K, Stitt M, Shin R (2011) Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes BMC systems. Biol 5:192

    CAS  Google Scholar 

  • Finneman J, Schjoerring JK (2000) Post-translational regulation of cytosolic glutamine synthetase genes by reversible phosphorylation and 14-3-3 protein interaction. Plant J 24:171–181

    Article  Google Scholar 

  • Gallie DR (2002) The 5′-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. Nucl Acids Res 30:3401–3411

    Article  PubMed  CAS  Google Scholar 

  • Gray F, Tirabassi R, Meyers H, Wu G, McWeeney S, Hook L, Nelson JA (2010) A viral microRNA down-regulates multiple cell cycle genes through mRNA 5′UTRs. PLoS Pathog 6:e1000967

    Article  Google Scholar 

  • Hamilton TL, Stoneley Sprigs KA, Bushell M (2006) TOPs and their regulation. Biochem Soc Trans 34:12–16

    Article  PubMed  CAS  Google Scholar 

  • Hulzink RJM, de Groot PFM, Croes AF, Quaedvlieg W, Twell D, Wullems GJ, van Herpen MMA (2002) The 5′-Untranslated region of the ntp303 gene strongly enhances translation during pollen tube growth, but not during pollen maturation. Plant Physiol 129:342–353

    Article  PubMed  CAS  Google Scholar 

  • Jaeger JA, Turner DH, Zuker M (1989) Improved predictions of secondary structures for RNA. Proc Natl Acad Sci USA 86:7706–7710

    Article  PubMed  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jiang P, Ninfa AJ (2007) The Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro. Biochemistry 37:12802–12810

    Article  Google Scholar 

  • Jiménez-López S, Mancera-Martínez E, Donayre-Torres A, Rangel C, Uribe L, March S, Jiménez-Sánchez G, Sánchez de Jiménez E (2011) Expression profile of maize (Zea mays L.) embryonic axes during germination: translational regulation of ribosomal protein mRNAs. Plant Cell Physiol 52:1719–1733

    Article  PubMed  Google Scholar 

  • Kawaguchi R, Bailey-Serres J (2002) Regulation of translational initiation in plants. Curr Opin Plant Biol 5:460–465

    Article  PubMed  CAS  Google Scholar 

  • Kawaguchi R, Bailey-Serres J (2005) mRNA sequence features that contribute to translational regulation in Arabidopsis. Nucl Acids Res 33:955–965

    Article  PubMed  CAS  Google Scholar 

  • Komarova AV, Tchufistova LS, Supina EV, Boni IV (2002) Protein S1 counteracts the inhibitory effect of the extended Shine–Dalgarno sequence on translation. RNA 8:137–1147

    Article  Google Scholar 

  • Koscianska E, Kalantidis K, Wypijewski K, Sadowski J, Tabler M (2005) Analysis of RNA silencing in agroinfiltrated leaves of Nicotiana benthamiana and Nicotiana tabaccum. Plant Mol Biol 59:647–661

    Article  PubMed  CAS  Google Scholar 

  • Kulkarni SD, Muralidharan B, Panda AC, Bakthavachalu B, Vindu A, Seshadri V (2011) Glucose-stimulated translation regulation of insulin by the 5′UTR-binding proteins. J Biol Chem 286:14146–14156

    Article  PubMed  CAS  Google Scholar 

  • Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y (1993) Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90:3009–3013

    Article  PubMed  CAS  Google Scholar 

  • Leckie BM, Stewart CN (2011) Agroinfiltration as a technique for rapid assays for evaluating candidate insect resistance transgenes in plants. Plant Cell Rep 30:325–334

    Article  PubMed  CAS  Google Scholar 

  • Lee K, Holland-Staley A, Cunninham PR (1996) Genetic analysis of the Shine–Dalgarno interaction: selection of alternative functional mRNA–rRNA combinations. RNA 2:1270–1285

    PubMed  CAS  Google Scholar 

  • Leigh JA, Dodsworth JA (2007) Nitrogen regulation in bacteria and Archaea. Annu Rev Microbiol 61:349–377

    Article  PubMed  CAS  Google Scholar 

  • Lima L, Seabra A, Melo P, Cullimore J, Carvalho H (2006) Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula. J Exp Bot 57:2751–2767

    Article  PubMed  CAS  Google Scholar 

  • Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 10:307–318

    Article  PubMed  Google Scholar 

  • Markham NR, Zuker M (2008) UNAFold: software for nucleic acid folding and hybridization. In: Keith JM (ed) Bioinformatics, vol II. Structure, function and applications, in methods in molecular biology, vol 453, pp 3–31. Humana Press, Totowa, NJ

  • McCarthy JEG, Brimacombe R (1994) Prokaryotic translation: the interactive pathway leading to initiation. Trends in Genet 10:402–407

    Article  CAS  Google Scholar 

  • Mogridge J, Greenblatt J (1998) Specific binding of Escherichia coli ribosomal protein S1 to boxA transcriptional terminator RNA. J Bacteriol 180:2248–2252

    PubMed  CAS  Google Scholar 

  • Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol 51:328–332

    Article  PubMed  CAS  Google Scholar 

  • Nakamoto T (2009) Evolution and the universality of the mechanism of initiation of protein synthesis. Gene 432:1–6

    Article  PubMed  CAS  Google Scholar 

  • Nakamoto T (2011) Mechanisms of the initiation of protein synthesis: in reading frame binding of ribosomes to mRNA. Mol Biol Rep 38:847–855

    Article  PubMed  CAS  Google Scholar 

  • Ninfa AJ, Jiang P (2005) PII signal transduction proteins: sensors of α-ketoglutarate that regulate nitrogen metabolism. Curr Opin Microbiol 8:168–173

    Article  PubMed  CAS  Google Scholar 

  • Nunes-Nesi A, Fernie AR, Stitt M (2010) Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Mol Plant 3:973–996

    Article  PubMed  CAS  Google Scholar 

  • Orom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30:460–471

    Article  PubMed  Google Scholar 

  • Ortega JL, Roche D, Sengupta-Gopalan C (1999) Oxidative turnover of soybean root glutamine synthetase: in vitro and in vivo studies. Plant Physiol 119:1483–1495

    Article  PubMed  CAS  Google Scholar 

  • Ortega JL, Temple SJ, Bagga S, Sengupta-Gopalan C (2001) Constitutive over-expression of cytosolic glutamine synthetase (GS1) genes in transgenic alfalfa demonstrates that GS1 may be regulated both at the level of RNA and protein turnover. Plant Physiol 126:109–121

    Article  PubMed  CAS  Google Scholar 

  • Ortega JL, Moguel-Esponda S, Potenza C, Conklin CF, Quintana A, Sengupta-Gopalan C (2006) The 3′ untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa. Plant J 45:842–846

    Article  Google Scholar 

  • Roberts CS, Rajagopal S, Smith LA, Nguyen TA, Yang W, Nugroho S, Ravi KS, Cao ML, Vijayachandra K, Patell V, Harcourt RL, Dransfield L, Desamero N, Slamet I, Keese P, Kilian A, Jefferson RA (1998) A comprehensive set of modular vectors for advanced manipulations and efficient transformation of plants by both Agrobacterium and direct DNA uptake methods. pCAMBIA vector release manual version 3.05. CAMBIA. Canberra, Australia

  • Rogers SG, Klee HJ, Horsch RB, Fraley RT (1987) Improved vectors for plant transformation: expression cassette vectors and new selectable markers. Methods Enzymol 153:253–277

    Article  CAS  Google Scholar 

  • Sakakibara H, Shimizu H, Hase T, Yamazaki Y, Takao T, Shimonishi Y, Sugiyama T (1996) Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots. J Biol Chem 271:29561–29568

    Article  PubMed  CAS  Google Scholar 

  • Satoh J, Kato K, Shinmyo A (2004) The 5′-untranslated region of the tobacco alcohol dehydrogenase gene function as an effective translational enhancer in plant. J Biosci Bioeng 98:1–8

    PubMed  CAS  Google Scholar 

  • Seabra AR, Vieira CP, Cullimore JV, Carvalho HG (2010) Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds. BMC Plant Biol 10:183

    PubMed  Google Scholar 

  • Simon B, Sengupta-Gopalan C (2010) The 3′ untranslated region of the two cytosolic glutamine synthetase (GS1) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine. Planta 232:1151–1162

    Article  PubMed  CAS  Google Scholar 

  • Smith CA, Weljie AM, Moorhead GBG (2003) Molecular properties of the putative nitrogen sensor PII from Arabidopsis thaliana. Plant J 33:353–360

    Article  PubMed  CAS  Google Scholar 

  • Stupina VA, Yuan X, Meskauskas A, Dinman JD, Simon AE (2011) Ribosome binding to a 5′ translational enhancer is altered in the presence of the 3′ untranslated region in Cap-independent translation of Turnip Crinkle Virus. J Virol 85:4638–4653

    Article  PubMed  CAS  Google Scholar 

  • Tzeng TY, Kong LR, Chen CH, Shaw CC, Yang CH (2009) Overexpression of the lily p70s6k gene in Arabidopsis affects elongation of flower organs and indicator TOR-dependent regulation of AP3, PI and SUP translation. Plant Cell Physiol 50:1695–1709

    Article  PubMed  CAS  Google Scholar 

  • Wang CT, Xu YN (2010) The 5′ untranslated region of the FAD3 mRNA is required for its translational enhancement at low temperature in Arabidopsis roots. Plant Sci 179:234–240

    Article  CAS  Google Scholar 

  • Wang R, Guan P, Chen M, Xing X, Zhang Y, Crawford NM (2010) Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer. Plant Physiol 154:423–432

    Article  PubMed  CAS  Google Scholar 

  • Zou Z, Eibil C, Koop HU (2003) The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency. Mol Genet Genomics 269:340–349

    Article  PubMed  CAS  Google Scholar 

  • Zuker M (1989) On finding all suboptimal foldings of an RNA molecule. Science 244:48–52

    Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health (Grant number S06 GM08136-32), by the US Department of Agriculture (Grant number 2007-03596), and by the Agricultural Experimental Station at New Mexico State University. We thank Dr. Suman Bagga and Dr. Laura Rodriguez-Uribe for their helpful comments on the manuscript.

Author information

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM, 88003, USA

    Jose Luis Ortega & Champa Sengupta-Gopalan

  2. Molecular Biology Graduate Program, New Mexico State University, Las Cruces, NM, 88003, USA

    Olivia L. Wilson

Authors
  1. Jose Luis Ortega
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Olivia L. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Champa Sengupta-Gopalan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Champa Sengupta-Gopalan.

Additional information

Communicated by H. Ronne.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ortega, J.L., Wilson, O.L. & Sengupta-Gopalan, C. The 5′ untranslated region of the soybean cytosolic glutamine synthetase β1 gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants. Mol Genet Genomics 287, 881–893 (2012). https://doi.org/10.1007/s00438-012-0724-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-012-0724-6

Keywords

Search

Navigation