Skip to main content
SpringerLink
Log in

The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency

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

Abstract

Regulation of chloroplast gene expression involves networked and concerted interactions of nucleus-encoded factors with their target sites on untranslated regions (UTRs) of chloroplast transcripts. So far, only a few cis -acting elements within such 5′UTR sequences have been identified as functional determinants of mRNA stability and efficient translation in Chlamydomonas in vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5′UTRs of tobacco psbA and rbcL fused to the coding region of the reporter gene uidA. Various mutant versions of the psbA leader, as well as rbcL/psbA hybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidA mRNA levels and a 1.5- to 6-fold reduction in uidA translation efficiency in all psbA 5′UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA 5′UTR stem-loop are required for mRNA stabilisation and translation. The 5′-terminal segment of the rbcL 5′UTR did not enhance the stability or translational activity of chimeric uidA mRNA under the standard light-dark regime of 16 h light and 8 h dark. Stabilising effects were, however, observed when the cells were kept continuously in the dark. Possible reasons for the influence of the 5′UTR of the tobacco psbA on mRNA stability and translation efficiency are discussed.

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. 2A, B.
Fig. 3A–C.
Fig. 4A–D.
Fig. 5A–D.

Similar content being viewed by others

References

  • Anthonisen IL, Salvador ML, Klein U (2001) Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas r einhardtii. RNA 7:1024–1033

    Article  CAS  PubMed  Google Scholar 

  • Barkan A (1993) Nuclear mutants of maize with defects in chloroplast polysome assembly have altered chloroplast metabolism. Plant Cell 5:389–402

    Google Scholar 

  • Barkan A, Goldschmidt-Clermont M (2000) Participation of nuclear genes in chloroplast gene expression. Biochimie 82:559–572

    CAS  PubMed  Google Scholar 

  • Bhattacharya D, Medlin L (1998) Algal phylogeny and the origin of land plants. Plant Physiol 116:9–15

    Article  CAS  Google Scholar 

  • Bogorad L (2000) Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol 18:257–263

    Article  CAS  PubMed  Google Scholar 

  • Danon A, Mayfield SP (1991) Light regulated translational activators: identification of chloroplast gene specific mRNA binding proteins. EMBO J 10:3993–4001

    CAS  PubMed  Google Scholar 

  • Drager RG, Zeidler M, Simpson CL, Stern DB (1996) A chloroplast transcript lacking the 3′ inverted repeat is degraded by 3′→5′ exoribonuclease activity. RNA 2:652–663

    CAS  PubMed  Google Scholar 

  • Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU (1999) In vivo analysis of plastid psbA rbcL and rpl32UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J 19:333–345

    Article  CAS  PubMed  Google Scholar 

  • Fargo DC, Boynton JE, Gillham NW (1999) Mutations altering the predicted secondary structure of a chloroplast 5′ untranslated region affect its physical and biochemical properties as well as its ability to promote translation of reporter mRNAs both in the Chlamydomonas reinhardtii chloroplast and in Escherichia coli. Mol Cell Biol 19:6980–6990

    CAS  PubMed  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    CAS  PubMed  Google Scholar 

  • Gillham NW, Boynton JE, Hauser CR (1994) Translational regulation of gene expression in chloroplasts and mitochondria. Annu Rev Genet 28:71–93

    CAS  PubMed  Google Scholar 

  • Hanley-Bowdoin L, Orozco EM Jr, Chua NH (1985) In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 15-bisphosphate carboxylase large subunit gene. Mol Cell Biol 5:2733–2745

    CAS  PubMed  Google Scholar 

  • Hauser CR, Gillham NW, Boynton JE (1998) Regulation of chloroplast translation. In: Rochaix JD, Goldschmidt-Clermont M, Merchant S (eds) The Molecular biology of chloroplasts and mitochondria in Chlamydomonas. Kluwer Academic Publishers, Netherlands, pp 197–217

  • Higgs DC, Shapiro RS, Kindle KL, Stern DB (1999) Small cis -acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation. Mol Cell Biol 19:8479–8491

    CAS  PubMed  Google Scholar 

  • Hirose T, Sugiura M (1996) Cis -acting elements and trans -acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts. EMBO J 15:1687–1695

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Kim J, Mullet JE (1994) Ribosome-binding sites on chloroplast rbcL and psbA mRNAs and light-induced initiation of D1 translation. Plant Mol Biol 25:437–448

    CAS  PubMed  Google Scholar 

  • Kim M, Christopher DA, Mullet JE (1993) Direct evidence for selective modulation of psbA,r poA, rbcL and 16S RNA stability during barley chloroplast development. Plant Mol Biol 22:447–463

    CAS  PubMed  Google Scholar 

  • Klein RR, Mason HS, Mullet JE (1988) Light-regulated translation of chloroplast proteins. I. Transcripts of psaA - psaB psbA and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings. J Cell Biol 106:289–301

    PubMed  Google Scholar 

  • Koop HU, Steinmüller K, Wagner H, Rössler C, Eibl C, Sacher L (1996) Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199:193–201

    CAS  PubMed  Google Scholar 

  • Mayfield SP, Cohen A, Danon A, Yohn CB (1994) Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5′ untranslated region. J Cell Biol 127:1537–1545

    CAS  PubMed  Google Scholar 

  • Mayfield SP, Yohn CB, Cohen A, Danon A (1995) Regulation of chloroplast gene expression. Annu Rev Plant Physiol Plant Mol Biol 46:147–166

    CAS  Google Scholar 

  • Monde RA, Schuster G, Stern DB (2000) Processing and degradation of chloroplast mRNA. Biochimie 82:573–582

    CAS  Google Scholar 

  • Nakamura T, Ohta M, Sugiura M, Sugita M (1999) Chloroplast ribonucleoproteins are associated with both mRNAs and intron-containing precursor tRNAs. FEBS Lett 276:437–441

    Article  Google Scholar 

  • Nakamura T, Ohta M, Sugiura M, Sugita M (2001) Chloroplast ribonucleoproteins function as a stabilizing factor of ribosome-free mRNAs in the stroma. J Biol Chem 276:147–152

    Article  CAS  PubMed  Google Scholar 

  • Mullet JE, Orozco EM, Chua NH (1995) Multiple transcripts for higher plant rbcL and aptB genes and localization of the transcription initiation site of the rbcL gene. Plant Mol Biol 4:39–54

    Google Scholar 

  • Nickelsen J, Fleischmann M, Boudreau E, Rahire M, Rochaix JD (1999) Identification of cis -acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas. Plant Cell 11:957–970

    Google Scholar 

  • Rochaix JD (1996) Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas r einhardtii. Plant Mol Biol 32:327–341

    CAS  PubMed  Google Scholar 

  • Rochaix JD, Kuchka M, Mayfield S, Schirmer-Rahire M, Girard-Bascou J, Bennoun P (1989) Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii. EMBO J 8:1013–1021

    CAS  PubMed  Google Scholar 

  • Rott R, Drager RG, Stern DB, Schuster G (1996) The 3′ untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators. Mol Gen Genet 252:676–683

    PubMed  Google Scholar 

  • Ruf S, Hermann M, Berger IJ, Carrer H, Böck R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19:870–875

    CAS  PubMed  Google Scholar 

  • Salvador ML, Klein U, Bogorad L (1993) Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas.Regulation by transcription and RNA degradation. Plant J 3:213–219

    CAS  PubMed  Google Scholar 

  • Sambrook ML, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Shen Y, Danon A, Christopher DA (2001) RNA binding-proteins interact specifically with the Arabidopsis chloroplast psbA mRNA 5′ untranslated region in a redox-dependent manner. Plant Cell Physiol 42:1071–1078

    CAS  PubMed  Google Scholar 

  • Shiina T, Allison L, Maliga P (1998) rbcL transcript levels in tobacco plastids are independent of light: reduced dark transcription rate is compensated by increased mRNA stability. Plant Cell 10:1713–1722

    CAS  PubMed  Google Scholar 

  • Singh M, Boutanaev A, Zucchi P, Bogorad L (2001) Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts. Proc Natl Acad Sci USA 98:2289–2294

    Article  CAS  PubMed  Google Scholar 

  • Stern DB, Gruissem W (1987) Control of plastid gene expression: 3′ inverted repeats act as mRNA processing and stabilising elements but do not terminate transcription. Cell 51:1145–1157

    CAS  PubMed  Google Scholar 

  • Stern DB, Radwanski ER, Kindle KL (1991) A 3′ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell 3:285–297

    Google Scholar 

  • Sugita M, Sugiura M (1996) Regulation of gene expression in chloroplasts of higher plants. Plant Mol Biol 32:315–326

    CAS  PubMed  Google Scholar 

  • Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917

    CAS  PubMed  Google Scholar 

  • Vaistij FE, Goldschmidt-Clermont M, Wostrikoff K, Rochaix JD (2000) Stability determinants in the chloroplast psbB/T/H mRNAs of Chlamydomonas r einhardtii. Plant J 21:469–482

    CAS  PubMed  Google Scholar 

  • Zerges W (2000) Translation in chloroplasts. Biochimie 82:583–601

    CAS  Google Scholar 

  • Zerges W, Girard-Bascou J, Rochaix JD (1997) Translation of the chloroplast psbC mRNA is controlled by interactions between its 5′ leader and the nuclear loci TBC1and TBC3in Chlamydomonas r einhardtii. Mol Cell Biol 17:3440–3448

    CAS  PubMed  Google Scholar 

  • Zuker M (1994) Prediction of RNA secondary structure by energy minimisation. Methods Mol Biol 25:267–294

    CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by the Deutsche Forschungsgemeinschaft (Ko 632-13/1,2). We are grateful to Prof. John Mullet (Texas A and M University, College Station, Texas, USA) for his unpublished data and valuable suggestions on this work

Author information

Authors and Affiliations

  1. Botanisches Institut, Ludwig-Maximilians-Universität, Menzinger Strasse 67, 80638, Munich, Germany

    Z. Zou, C. Eibl & H.-U. Koop

Authors
  1. Z. Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Eibl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H.-U. Koop
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H.-U. Koop.

Additional information

Communicatred by R. G. Herrmann

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zou, Z., Eibl, C. & Koop, HU. The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency. Mol Gen Genomics 269, 340–349 (2003). https://doi.org/10.1007/s00438-003-0842-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-003-0842-2

Keywords

Search

Navigation