Streptomyces atp nucleotide 3′-pyrophosphokinase–gene cloning and sequence analysis

Streptomyces ATP nucleotide 3′-pyrophosphokinase is an extracellular enzyme that transfers 5′-β,γ-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3′-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.