Isolation of Streptomyces lividans ribosomes and initiation factors and their characterization using in vitro mRNA binding assays

A primer extension inhibition (toeprint) assay was developed using ribosomes and ribosomal subunits from Streptomyces lividans. This assay allowed the study of ribosome binding to streptomycete leaderless and leadered mRNA. Purified 30S subunits were unable to form a ternary complex on aph or cat leaderless mRNAs, whereas 70S ribosomes could form ternary complexes on both of these mRNAs. 30S subunits formed ternary complexes on aph and malE leadered mRNA. The translation initiation factors (IF1, IF2, IF3) from S. lividans were isolated and included in toeprint and filter binding assays with leadered and leaderless mRNA. Generally, the IFs reduced the toeprint signal on leadered mRNA; however, incubation of IF1 and IF2 with high-salt washed 30S subunits promoted the formation of a ternary complex on aph leaderless mRNA. Our data suggest that, as reported for Escherichia coli, initiation complexes with leaderless mRNAs might use a novel pathway involving 70S ribosomes or 30S subunits bound by IF1 and IF2 but not IF3. Some mRNA-ribosome-initiator tRNA reactions that yielded weak or no toeprint signals still formed ternary complexes in filter binding assays, suggesting the occurrence of ternary complex interactions that are not stable in the toeprint assay. Further, the streptomycete 50S subunit was observed forming a ternary complex on aph and cat leaderless mRNAs; a model is presented for an alternate translation initiation pathway involving 50S subunits binding to leaderless mRNA in the streptomycetes. This is the first comprehensive look at the interactions of the molecular components involved in translation in the streptomycetes.