Summary
In vitro transcripts from the 3′ flanking regions of mustard chloroplast genes were tested for protein binding in a chloroplast extract. Efficient and sequence-specific RNA-protein interaction was detected with transcripts of the genes trnK, rps16 and trnH, but not with the 3′ terminal region of trnQ RNA. The trans-acting component required for specific complex formation is probably a single 54 kDa polypeptide. The protein-binding region of the rps16 3′ terminal region was mapped and compared with that of the trnK transcript determined previously. Both regions reveal a conserved 7-mer UUUAUCU followed by a stretch of U residues. Deletion of the trnK 3′ U cluster resulted in more than 80% reduction in the binding activity, and after deletion of both the U stretch and the 7-mer motif no binding at all was detectable. RNase protection experiments indicate that the protein-binding regions of both the rps16 and trnK transcripts correlate with the positions of in vivo 3′ ends, suggesting an essential role for the 54 kDa binding protein in RNA 3′ end formation. In the case of the trnK gene, evidence was obtained for read-through transcripts that extend into the psbA coding region, thus pointing to the possibility of trnK-psbA cotranscription.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adams CC, Stern DB (1990) Control of mRNA stability in chloroplasts by 3′ inverted repeats: effects of stem and loop mutations on degradation of psbA mRNA in vitro. Nucleic Acids Res 18:6003–6010
Boyer SK, Mullet JE (1986) Characterization of P. sativum chloroplast psbA transcripts produced in vivo, in vitro and in E. coli. Plant Mol Biol 6:229–244
Callahan FE, Ghirardi ML, Sopory SK, Mehta AM, Edelman M, Mattoo AK (1990) A novel metabolic from of the 32k Da-D1 protein in the grana-localized reaction center of photosystem II. J Biol Chem 256:15357–15360
Chen LJ, Orozco EM (1988) Recognition of prokaryotic terminators by spinach chloroplast RNA polymerase. Nucleic Acids Res 16:8411–8431
Crossland LD, Rodermel SR, Bogorad L (1984) Single gene for the large subunit of ribulosebisphosphate carboxylase in maize yields two differentially regulated mRNAs. Proc Natl Acad Sci USA 81:4060–4064
Deng XW, Gruissem W (1987) Control of plastid gene expression during development: The limited role of transcriptional regulation. Cell 49:379–387
Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interaction by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525
Fromm H, Devic M, Fluhr R, Edelman M (1985) Control of psbA gene expression: In mature chloroplasts, light regulation of 32 kDa protein synthesis is independent of transcript level. EMBO J 4:291–295
Gamble PE, Mullet JE (1989) Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley. J Biol Chem 264:7236–7243
Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9:3047–3060
Greenberg JR (1979) Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res 6:715–732
Gruissem W (1989) Chloroplast gene expression: how plants turn their plastids on. Cell 56:161–170
Haley J, Bogorad L (1990) Alternative promoters are used for genes within maize chloroplast polycistronic transcription units. Plant Cell 2:323–333
Hanley-Bowdoin L, Orozco EM, Chua NH (1985) Transcription of chloroplast genes by homologous and heterologous RNA polymerases. In: Steinback KE, Bonitz S, Arntzen CJ, Bogorad L (eds) Molecular biology of the photosynthetic apparatus. Cold Spring Harbor Laboratory Press, New York, pp 311–318
Henikoff S (1987) Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol 155:156–165
Higgins CF, McLaren RS, Newbury SF (1988) Repetitive extragenic polindromic sequences, mRNA stability and gene expression: evolution by gene conversion? Gene 72:3–14
Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun C-R, Meng B-Y, Li Y-Q, 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 inversion during the evolution of cereals. Mol Gen Genet 217:185–194
Klein RR, Mullet JE (1986) Regulation of chloroplast-encoded chlorophyll-binding protein translation during higher plant chloroplast biogenesis. J Biol Chem 261:11138–11145
Klein RR, Mullet JE (1987) Control of gene expression during higher plant chloroplast biogenesis. J Biol Chem 9:4341–4348
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Leibold EA, Munro HN (1988) Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5′ untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Natl Acad Sci USA 85:2171–2175
Li Y, Sugiura M (1990) Three distinct ribonucleoproteins from tobacco chloroplasts: each contains a unique amino terminal acidic domain and two ribonucleoprotein consensus motifs. EMBO J 9:3059–3066
Link G (1984) Hybridization study of developmental plastid gene expression in mustard (Sinapis alba L.) with cloned probes for most plastid DNA regions. Plant Mol Biol 3:243–248
Link G, Langridge U (1984) Structure of the chloroplast gene for the precursor of the Mr 32000 photosystem II protein from mustard (Sinapis alba). Nucleic Acids Res 12:945–957
Marion-Poll A, Hibbert CS, Radebaugh CA, Hallick RB (1988) Processing of mono-, di- and tricistronic transfer RNAs precursors in a spinach or pea chloroplast soluble extract. Plant Mol Biol 11:45–56
Mullet JE, Orozco EM, Chua N-H (1985) Multiple transcripts for higher plant rbcL and atpB genes and localization of the transcription initiation site of the rbcL gene. Plant Mol Biol 4:39–54
Neuhaus H (1989) Nucleotide sequence of the chloroplast genes for tRNAGln and the 4 kD K polypeptide of photosystem II from mustard (Sinapis alba). Nucleic Acids Res 17:444
Neuhaus H, Link G (1987) The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide. Curr Genet 11:251–257
Neuhaus H, Link G (1990) The chloroplast psbK operon from mustard (Sinapis alba L.): multiple transcripts during seedling development and evidence for divergent overlapping transcription. Curr Genet 18:377–383
Neuhaus H, Scholz A, Link G (1989) Structure and expression of a split chloroplast gene from mustard (Sinapis alba): ribosomal protein gene rps16 reveals unusual transcriptiona features and complex RNA maturation. Curr Genet 15:63–70
Nickelsen J, Link G (1989) Interaction of a 3′ RNA region of the mustard trnK gene with chloroplast proteins. Nucleic Acids Res 17:9637–9648
Nickelsen J, Link G (1990) Nucleotide sequence of the mustard chloroplast genes trnH and rps19′. Nucleic Acids Res 18:1051
Ohyama K, Fukuzawa H, Kohchi T, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S-I, Inokuchi H, Ozeki H (1986) Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322:572–574
Poulsen C (1984) Two mRNA species differeing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene. Carlsberg Res Commun 48:57–80
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
Rock CD, Barkan A, Taylor WC (1987) The maize plastid psbB-psbF-petB-petD gene cluster: spliced and unspliced petB and petD RNAs encode alternative products. Curr Genet 12:69–77
Ruf M, Kössel H (1988) Structure and expression of the gene coding for the alpha-subunit of DNA-dependent RNA polymerase from the chloroplast genome of Zea mays. Nucleic Acids Res 16:5741–5753
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, New York
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 B-Y, 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
Stern DB, Gruissem W (1987) Control of plastid gene expression: 3′ inverted repeats acts as mRNA processing and stabilizing elements, but do not terminate transcription. Cell 51:1145–1157
Stern DB, Gruissem W (1989) Chloroplast mRNA 3′ end maturation is biochemically distinct from prokaryotic mRNA processing. Plant Mol Biol 13:615–625
Stern DB, Jones H, Gruissem W (1989) Function of plastid mRNA 3′ inverted repeats. RNA stabilization and genespecific protein binding. J Biol Chem 264:18742–18750
Sugita M, Shinozaki K, Sugiura M (1985) Tobacco chloroplast tRNALys(UUU) genes contain a 2.5 kilobase pair intron: An open reading frame and a conserved boundary sequence in the intron. Proc Nat Acad Scu USA 82:3557–3561
Sugiura M (1989) The chloroplast genome. In: Marcus A (ed) The biochemistry of plants, vol 15. Academic Press, New York, pp 133–150
Tanaka M, Obokata J, Chunwongse J, Shinozaki K, Suguira 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
Thomas F, Zeng GQ, Mache R, Briat JF (1988) Transcription study of the genes encoded in the region of the junction between the large single copy and the inverted repeat A of spinach chlooroplast DNA. Plant Mol Biol 10:447–457
Wang MJ, Davis NW, Gegenheimer P (1988) Novel mechanisms for maturation of chloroplast transfer RNA precursors. EMBO J 7:1567–1574
Weil JH (1987) Organization and expression of the chloroplast genome. Plant Sci 49:149–157
Westhoff P, Herrmann RG (1988) Complex RNA maturation in chloroplasts. Eur J Biochem 171:551–564
Wilusz J, Shenk T (1988) A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell 52:221–228
Yamaguchi-Shinozaki K, Shinozaki K, Sugiura M (1987) Processing of precursor tRNAs in a chloroplast lysate. FEBS Lett 215:132–136
Author information
Authors and Affiliations
Additional information
Communicated by H. Saedler
Rights and permissions
About this article
Cite this article
Nickelsen, J., Link, G. RNA-protein interactions at transcript 3′ ends and evidence for trnK-psbA cotranscription in mustard chloroplasts. Molec. Gen. Genet. 228, 89–96 (1991). https://doi.org/10.1007/BF00282452
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00282452