Characterization of the chloroplast psbA and trnK genes

Abstract

As the plant proceeds through development from a seed to a photosynthetically competent organism, plastids of leaf tissue differentiate into chloroplasts. This process is due to the coordinated expression of nuclear and chloroplast genes. Chloroplast genes, which are contained on the multiple copies of the chloroplast genome, are expressed during light-induced chloroplast development. As a preliminary step in the investigation of chloroplast gene regulation, genes are isolated and characterized. In this manuscript, the characterization of the chloroplast psbA and trnK genes is described. The in vivo psbA transcripts, from Pisum sativum, were analyzed using a variety of molecular biology techniques. In vitro transcription of the gene, as well as its transcription in E. coli, demonstrated the importance of its procaryote-like promoter in the transcription of the psbA gene. After switching to Hordeum vulgare (due to its superiority over peas with respect to the study of plastid biogenesis) the stability of transcripts of the psbA gene was investigated. Increased stability of the psbA mRNA was noted in lysed plastid extracts (using in vitro synthesized RNAs), from illuminated plants as opposed to plastids isolated from dark grown plants. The increased stability of the psbA mRNA in illuminated tissue suggests that post-transcriptional regulation may be involved in psbA expression. Another possibility for the regulation of psbA gene expression which is consistent with previous findings was a translational regulatory scheme. This scheme involved the preferential expression of genes as determined by the codons present within their protein coding sequences. This hypothesis was investigated indirectly through the characterization of the trnK gene. The characterization of this gene revealed a large intron in the anticodon loop of the tRNA. Within the intron is a 506 amino acid open reading frame (URF) which shows some homology to yeast maturases. Detailed analysis of the transcripts indicated the possibility of a unique scenario for the regulation of this gene. Even though it appears that codon usage may not play in important role in the induction of psbA gene product synthesis, the trnK gene has proven to be interesting with respect to its own regulation.