A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMettRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.A novel class of antimicrobial agents, the oxazolidinones, target a wide spectrum of gram-positive and anaerobic bacteria (4, 6, 9, 28). These compounds act by inhibiting protein synthesis and have no effect on replication or transcription (8). Cell extracts exposed to oxazolidinones are impaired in protein synthesis when programmed by native mRNAs but do not appear to be inhibited when programmed by synthetic mRNAs that lack the signals required for initiation and termination of translation (7,8,26). This suggested that these compounds may target the initiation reaction. The oxazolidinone PNU-100766 (linezolid; Fig. 1) inhibits binding of the initiator fMettRNA to the 70S ribosomal particle programmed with a synthetic mRNA that harbors a Shine-Dalgarno sequence and a properly spaced initiation codon (29).Mutations to oxazolidinone resistance map to domain V of the 23S rRNA in Halobacterium halobium (18), Staphylococcus aureus (27), and the enterococci while mapping to domains IV and V in Escherichia coli (33). The position of these PNU-100766 resistance mutations suggested to us that the oxazolidinones may target peptidyl transferase indirectly by affecting the binding of the initiator tRNA. Since the P site accommodates the initiator tRNA and the nascent protein, these drugs could also affect the affinity of the peptidyl-tRNA for the ribosome.Recent studies have indicated that the oxazolidinones bind to 70S ribosomes, as well as to 50S subunits (19), but not to 30S subunits. In contrast, a report by Matassova et al. (20) demonstrated that oxazolidinone footprints map to the central domain of the 16S rRNA whereas the 23S rRNA ...