NUCLEAR-ENCODED CHLOROPLAST RIBOSOMAL-PROTEIN L27 OF NICOTIANA-TABACUM - CDNA SEQUENCE AND ANALYSIS OF MESSENGER-RNA AND GENES

A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector lambda-gt11. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19 641 precursor to an M(r) 14 420 mature protein which is homologous to Escherichia coli ribosomaL protein L27. One cDNA (L27-1;882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail. A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27-protein. The mature L27 protein has 66%, 61%, 56% and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearothermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7. The carboxyl end of tobacco L27 has an unusual cysteine-rich sequence (CFCCC) of unknown function. Northern blot analysis revealed a single band of mRNA corresponding in size (0.85-0.90 kb) to full-length L27 cDNAs. Two cDNAs for tobacco ribosomal protein L27 were identical in coding sequence, but differed in 3'-noncoding sequence. Hybridization of L27 cDNA probes to restriction enzyme digests of tobacco genomic DNA and to cloned genomic fragments provided additional evidence suggesting that more than one gene exists for tobacco L27.