A novel protein with DNA binding activity from tobacco chloroplast nucleoids

Abstract

A 41-kD DNA binding protein with a basic pl was purified from chloroplast nucleoids in photomixotrophically cultured tobacco cells, and its amino acid sequence was determined. Using this sequence information, its cDNA (CND41) was isolated, and its nucleotide sequence was determined. The predicted amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. A distinctive helix-turn-helix motif in the lysine-rich N-terminal region of the mature protein and an aspartyl protease active site motif were predicted. Expression of a series of truncated CND41 proteins in Escherichia coli indicated that the lysine-rich region is essential for DNA binding and that CND41 nonspecifically binds chloroplast DNA. Protein gel blot analyses showed CND41 mainly in cells and/or tissues containing nonphotosynthesizing, actively growing plastids. In addition, the accumulation of chloroplast transcripts in these cells and/or tissues (e.g., transcripts for QB binding protein of photosystem II [psbA] and large subunit of ribulose bisphosphate carboxylase [rbcL]) was negatively correlated with the accumulation of CND41. Analyses of cultured cells of transgenic tobacco with reduced CND41 levels showed a higher level of expression of chloroplast genes compared with that of the wild type. We discuss the possible function of CND41 as a negative regulator of chloroplast gene expression.