Overexpression of the clpP 5'-untranslated region in a chimeric context causes a mutant phenotype, suggesting competition for a clpP-specific RNA maturation factor in tobacco chloroplasts.
The plastid ribosomal RNA (rrn) operon promoter was fused with DNA segments encoding the leader sequence (5'-untranslated region [UTR]) of plastid mRNAs to compare their efficiency in mediating translation of a bacterial protein neomycin phosphotransferase (NPTII) in tobacco (Nicotiana tabacum) chloroplasts. In young leaves, NPTII accumulated at 0.26% and 0.8% of the total soluble leaf protein from genes with the clpP and atpB 5'-UTR, respectively. Interestingly, expression of NPTII from the promoter with the clpP 5'-UTR (0.26% NPTII) caused a mutant (chlorotic) phenotype, whereas plants accumulating approximately 0.8% NPTII from the atpB 5'-UTR were normal green, indicating that the mutant phenotype was independent of NPTII accumulation. Low levels of monocistronic clpP mRNA and accumulation of intron-containing clpP transcripts in the chlorotic leaves suggest competition between the clpP 5'-UTR in the chimeric transcript and the native clpP pre-mRNA (ratio 16:1) for an mRNA maturation factor. Because maturation of 11 other intron-containing mRNAs was unaffected in the chlorotic leaves, it appears that the factor is clpP specific. The mutant phenotype is correlated with reduced levels (approximately 2 times) of the ClpP1 protease subunit, supporting an important role for ClpP1 in chloroplast development.
Waksman Institute, 190 Frelinghuysen Road, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020, USA.
Hiroshi
H
Kuroda
Pal
P
Maliga
D020121:5' Untranslated Regions / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics; D049071:Endopeptidase Clp; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010860:Pigments, Biological / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis; D020871:RNA Stability; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
D020121:5' Untranslated Regions; D004274:DNA, Recombinant; D010860:Pigments, Biological; D018749:RNA, Plant; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases