Chloroplast ribosomal protein L12 is encoded in the nucleus: construction and identification of its cDNA clones and nucleotide sequence including the transit peptide.
An architectural feature found in all classes of ribosomes is a thin, 10-nm-long protuberance in the large subunit, generated by multiple copies of r-protein L12. The primary structure of spinach chloroplast r-protein L12 is known [Bartsch, M., Kimura, M., & Subramanian, A. R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6871-6875], but the location of its gene, whether in the organelle or in the nucleus, has not been determined. Therefore, we synthesized four oligodeoxynucleotides based on the amino acid sequence data and used them to probe a spinach cDNA library we constructed in lambda gt11 vector. cDNA inserts from four of the hybridizing recombinant clones were characterized and sequenced. The data showed that they are reverse transcripts of varying length, all derived from a single poly(A+) RNA species. The longest cDNA molecule is 900 base pairs (bp) long and includes a 5' noncoding sequence followed by two neighboring AUG codons both in the consensus, eukaryotic initiator context, a 56-codon-long transit peptide sequence (starting from the first AUG codon), the amino acid sequence of mature L12 protein, and a 238 bp long 3' downstream noncoding sequence including a polyadenylation signal and the start of the poly(A) tail. The transit peptide sequence has an unusual amino acid composition similar to that of other known chloroplast transit peptides. Northern blot analysis of the poly(A+) RNA isolated from spinach seedlings and probed with the cDNA insert revealed the occurrence of a strong, broad, 950-nucleotide-long band of the corresponding poly(A+)-containing mRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)
Giese
K
K
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, West Germany.
Subramanian
A R
AR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics; D011061:Poly A / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics*
D003062:Codon; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D011061:Poly A; C016700:ribosomal protein L7-L12; D004247:DNA