Molecular characterization of full-length cDNAs for nuclear-encoded chloroplast ribosomal proteins L12, L24 and L27 of Nicotiana tabacum.

Abstract

Poly(A)⁺ mRNA isolated from Nicotiana tabacllm (cv. Petite havana) leaves was used to prepare a cDNA library in the expression vector Agtll. The library was screened with antibodies raised against proteins of chloroplast 50S ribosomal subunits. Full length cDNAs for three nuclear-encoded tobacco chloroplast large ribosomal subunit proteins were identified and sequenced. The deduced protein sequences are homologous to E. coli ribosomal proteins L12 (involved in GTP hydrolysis), L24 (a ribosomal RNA binding protein) and L27 (a constituent of peptidyltransferase ). The full-length cDNA encoding tobacco chloroplast ribosomal protein L12 is 773 nucleotides long, and encodes a mature protein of 133 amino acids and a transit peptide of 53 amino acids. The amino terminus of the mature protein was confirmed by protein sequencing of the first 12 amino acids. The deduced amino acid sequence of tobacco L12 protein has a high degree of identity with chloroplast ribosomal protein L12 of spinach (70%) and E. coli (51 % ). A full-length cDNA encoding tobacco chloroplast ribosomal protein L24 is 862 nucleotides long and encodes a mature protein of 157 amino acids and a putative transit peptide of 30 amino acids. The deduced amino acid sequence of tobacco L24 protein has a high degree of identity with chloroplast ribosomal protein L24 of pea (73%) and E. coli (35%). The fusion protein from the clone coding the tobacco L24 ribosomal protein was bound to nitrocellulose filters and used as affinity matrix to purify the antibody to the L24 protein. Monospecific antibody to L24 was used to identify, by Western blotting, the L24 ribosomal protein among purified proteins of the large subunit of the chloroplast ribosome. A full-length eDNA encoding tobacco ribosomal protein L27 is 882 nucleotides long and encodes a mature protein of 128 amino acids and a putative transit peptide of 51 amino acids. The deduced amino acid sequence of tobacco L27 protein has a high degree of identity with E. coli ribosomal protein L27 (64%) in the overlapping region between the two proteins. Besides the transit peptide, both ribosomal proteins L24 and L27 sequences have a carboxyl-terminal extension. Using Northern blot analysis, unique full size cytoplasmic mRNAs were observed for the three different proteins. For each of the three proteins (L12, L24, L27), at least two cDNAs with identical coding sequence and different 3'-non-coding sequences were identified. Southern blot analysis of genomic DNA indicated the presence of more than one gene for each of the three proteins. Furthermore, genomic clones likely to represent two different genes were isolated for tobacco ribosomal protein L27.