Abstract
The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b 6 /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5′-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5′-UTRs, the psbN 5′-UTR has two processing sites, at −39 and −24 upstream from the initiation site. Processing at −39 enhanced the translation rate fivefold. In contrast, processing at −24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5′-UTR has no Shine–Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5′-UTR or with deleted psbN 5′-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5′-UTR. Mobility shift assays using 10 other chloroplast 5′-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.
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References
Adachi Y, Kuroda H, Yukawa Y, Sugiura M (2012) Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5′-processing and translational coupling. Nucleic Acids Res 40:3152–3158
Alexander C, Faber N, Klaff P (1998) Characterization of protein-binding to the spinach chloroplast psbA mRNA 5′ untranslated region. Nucleic Acids Res 26:2265–2272
Barkan A (1988) Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs. EMBO J 7:2637–2644
Bock R (2007) Structure, function, and inheritance of plastid genomes. In: Bock R (ed) Cell and molecular biology of plastids. Springer, Berlin Heidelberg, pp 29–63
Felder S, Meierhoff K, Sane AP, Meurer J, Driemel C, Plücken H, Klaff P, Stein B, Bechtold N, Westhoff P (2001) The nucleus-encoded HCF107 gene of Arabidopsis provides a link between intercistronic RNA processing and the accumulation of translation-competent psbH transcripts in chloroplasts. Plant Cell 13:2127–2141
Franzetti B, Carol P, Mache R (1992) Characterization and RNA-binding properties of a chloroplast S1-like ribosomal protein. J Biol Chem 267:19075–19081
Gómez SM, Nishio JN, Faull KF, Whitelegge JP (2002) The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry. Mol Cell Proteomics 1:46–59
Gruissem W, Tonkyn JC (1993) Control mechanisms of plastid gene expression. Crit Rev Plant Sci 12:19–55
Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun CR, Meng BY, Li YQ, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M (1989) The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet 217:185–194
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
Hirose T, Sugiura M (1997) Both RNA editing and RNA cleavage are required for translation of tobacco chloroplast ndhD mRNA: a possible regulatory mechanism for the expression of a chloroplast operon consisting of functionally unrelated genes. EMBO J 16:6804–6811
Hirose T, Sugiura M (2004a) Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs. Plant Cell Physiol 45:114–117
Hirose T, Sugiura M (2004b) Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence. Nucleic Acids Res 32:3503–3510
Ikeuchi M, Koike H, Inoue Y (1989) N-terminal sequencing of low-molecular-mass components in cyanobacterial photosystem II core complex. Two components correspond to unidentified open reading frames of plant chloroplast DNA. FEBS Lett 253:178–182
Kawaguchi H, Fukuda I, Shiina T, Toyoshima Y (1992) Dynamical behavior of psb gene transcripts in greening wheat seedlings. I. Time course of accumulation of the psbA through psbN gene transcripts during light-induced greening. Plant Mol Biol 20:695–704
Kohchi T, Yoshida T, Komano T, Ohyama K (1988) Divergent mRNA transcription in the chloroplast psbB operon. EMBO J 7:885–891
Krech K, Fu HY, Thiele W, Ruf S, Schöttler MA, Bock R (2013) Reverse genetics in complex multigene operons by co-transformation of the plastid genome and its application to the open reading frame previously designated psbN. Plant J 75:1062–1074
Kuroda H, Suzuki H, Kusumegi T, Hirose T, Yukawa Y, Sugiura M (2007) Translation of psbC mRNAs starts from the downstream GUG, not the upstream AUG, and requires the extended Shine-Dalgarno sequence in tobacco chloroplasts. Plant Cell Physiol 48:1374–1378
Marín-Navarro J, Manuell AL, Wu J, Mayfield SP (2007) Chloroplast translation regulation. Photosynth Res 94:359–374
Meng BY (1991) Studies on expression of photosynthesis-related genes in tobacco chloroplasts (in Japanese), Dissertation, Nagoya University, Japan
Nickelsen J (2003) Chloroplast RNA-binding proteins. Curr Genet 43:392–399
Peled-Zehavi H, Danon A (2007) Translation and translational regulation in chloroplasts. In: Bock R (ed) Cell and molecular biology of plastids. Springer-Verlag, Berlin Heidelberg, pp 249–281
Plöscher M, Granvogl B, Zoryan M, Reisinger V, Eichacker LA (2009) Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts. Proteomics 9:625–635
Prikryl J, Rojas M, Schuster G, Barkan A (2011) Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein. Proc Natl Acad Sci USA 108:415–420
Reinbothe S, Reinbothe C, Heintzen C, Seidenbecher C, Parthier B (1993) A methyl jasmonate-induced shift in the length of the 5′ untranslated region impairs translation of the plastid rbcL transcript in barley. EMBO J 12:1505–1512
Robida MD, Merhige PM, Hollingsworth MJ (2002) Proteins are shared among RNA-protein complexes that form in the 5′ untranslated regions of spinach chloroplast mRNAs. Curr Genet 41:53–62
Schmitz-Linneweber C, Small I (2008) Pentatricopeptide repeat proteins: a socket set for organelle gene expression. Trends Plant Sci 13:663–670
Schmitz-Linneweber C, Maier RM, Alcaraz JP, Cottet A, Herrmann RG, Mache R (2001) The plastid chromosome of spinach (Spinacia oleracea): complete nucleotide sequence and gene organization. Plant Mol Biol 45:307–315
Schmitz-Linneweber C, Williams-Carrier R, Barkan A (2005) RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5′ region of mRNAs whose translation it activates. Plant Cell 17:2791–2804
Sengupta J, Agrawal RK, Frank J (2001) Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA. Proc Natl Acad Sci USA 98:11991–11996
Shi LX, Schröder WP (2004) The low molecular mass subunits of the photosynthetic supracomplex, photosystem II. Biochim Biophys Acta 1608:75–96
Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5:2043–2049
Sørensen MA, Fricke J, Pedersen S (1998) Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo. J Mol Biol 280:561–569
Stern DB, Goldschmidt-Clermont M, Hanson MR (2010) Chloroplast RNA metabolism. Annu Rev Plant Biol 61:125–155
Subramanian AR (1983) Structure and functions of ribosomal protein S1. Prog Nucleic Acid Res Mol Biol 28:101–142
Sugiura M (1992) The chloroplast genome. Plant Mol Biol 19:149–168
Sugiura M (2014) Plastid mRNA translation. In: Maliga P (ed) Chloroplast biotechnology: methods and protocols, methods in molecular biology, vol 1132. Springer, New York, pp 73–91
Sugiura M, Hirose T, Sugita M (1998) Evolution and mechanism of translation in chloroplasts. Annu Rev Genet 32:437–459
Suzuki H, Kuroda H, Yukawa Y, Sugiura M (2011) The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB-atpE dicistronic mRNAs in chloroplasts. Nucleic Acids Res 39:9405–9412
Tanaka M, Obokata J, Chunwongse J, Shinozaki K, Sugiura M (1987) Rapid splicing and stepwise processing of a transcript from the psbB operon in tobacco chloroplasts: determination of the intron sites in petB and petD. Mol Gen Genet 209:427–431
Torabi S, Umate P, Manavski N, Plöchinger M, Kleinknecht L, Bogireddi H, Herrmann RG, Wanner G, Schröder WP, Meurer J (2014) PsbN is required for assembly of the photosytem II reaction center in Nicotiana tabacum. Plant Cell 26:1183–1199
Umena Y, Kawakami K, Shen JR, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473:55–60
Wakasugi T, Tsuzuki J, Ito S, Nakashima K, Tsuzuki T, Sugiura M (1994) Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii. Proc Natl Acad Sci USA 91:9794–9798
Westhoff P, Herrmann RG (1988) Complex RNA maturation in chloroplasts. The psbB operon from spinach. Eur J Biochem 171:551–564
Wobbe L, Schwarz C, Nickelsen J, Kruse O (2008) Translational control of photosynthetic gene expression in phototrophic eukaryotes. Physiol Plant 133:507–515
Yukawa M, Tsudzuki T, Sugiura M (2005) The 2005 version of the chloroplast DNA sequence from tobacco (Nicotiana tabacum). Plant Mol Biol Rep 23:359–365
Yukawa M, Kuroda H, Sugiura M (2007) A new in vitro translation system for non-radioactive assay from tobacco chloroplasts: effect of pre-mRNA processing on translation in vitro. Plant J 49:367–376
Zghidi W, Merendino L, Cottet A, Mache R, Lerbs-Mache S (2007) Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psbN gene in plastids. Nucleic Acids Res 35:455–464
Zhelyazkova P, Sharma CM, Förstner KU, Liere K, Vogel J, Börner T (2012) The primary transcriptome of barley chloroplasts: numerous noncoding RNAs and the dominating role of the plastid-encoded RNA polymerase. Plant Cell 24:123–136
Acknowledgments
We thank Prof. Yasushi Yukawa for providing us with laboratory space and equipment and Maki Yukawa for discussions. This work was supported by the New Energy and Industrial Technology Development Organization, and Grants-in-Aid for Scientific Research (19370021, 22570050 to M.S., 20570043 to H.K.).
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Kuroda, H., Sugiura, M. Processing of the 5′-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA. Plant Mol Biol 86, 585–593 (2014). https://doi.org/10.1007/s11103-014-0248-z
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DOI: https://doi.org/10.1007/s11103-014-0248-z