Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene
Gruissem, W.; Zurawski, G.
EMBO Journal 4(7): 1637-1644
1985
ISSN/ISBN: 0261-4189 PMID: 2992936 Document Number: 247306
A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements. Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNA species. Transcription of the spinach chloroplast tRNA2Met gene (trn M2) in vitro requires 5' upstream DNA sequences. Deletion of 5' DNA sequences with exonuclease Bal31 was used to establish the 5' boundary of the promoter region. This boundary is part of a DNA sequence with partial homology to the prokaryotic -35 region. Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic -10 region. Synthetic oligonucleotides were used fused to trn M2 5' deletion mutants to create insertions, deletions and base substitutions in these regions. Internal deletion mutants demonstrated that the -10 promoter element is also required for transcription in vitro. The arrangement of DNA sequences recognzied by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.