Evidence for a translation-mediated attenuation of a spinach chloroplast rDNA operon

Abstract

The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination. The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide. In vitro synchronized transcription by E. coli RNA polymerase shows that pauses do occur in the leader region. By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes. The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid. The resulting plasmids have been used to transform a GalK- E. coli strain. Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control. However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP. These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression.