The Pseudomonas aeruginosa Initiation Factor IF-2 Is Responsible for Formylation-independent Protein Initiation in P. aeruginosa

Formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNA(fMet). Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNA(fMet)) and Met-tRNA(fMet). The E. coli IF-2, which participates exclusively in formylation- dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNA(fMet) in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation- independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNA(fMet) in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNA(fMet) in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.