SUMMARY Capistruin, a ribosomally synthesized post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is unknown. Capistruin is a threaded-lasso peptide (lariat peptide), comprising an N-terminal 9-amino-acid ring followed by a 10-amino-acid C-terminal tail that is threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capustruin, like MccJ25, inhibits wild-type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25 due to a mutation in an RNAP subunit gene exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.

MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.

Gp39, a small protein encoded by Thermus thermophilus phage P23–45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

Transcription initiation complexes formed by bacterial RNA polymerases (RNAPs) exhibit dramatic species-specific differences in stability, leading to different strategies of transcription regulation. The molecular basis for this diversity is unclear. Promoter complexes formed by RNAP from Thermus aquaticus (Taq) are considerably less stable than Escherichia coli RNAP promoter complexes, particularly at temperatures below 37°C. Here, we used a fluorometric RNAP molecular beacon assay to discern partial RNAP-promoter interactions. We quantitatively compared the strength of E. coli and Taq RNAPs partial interactions with the −10, −35 and UP promoter elements; the TG motif of the extended −10 element; the discriminator and the downstream duplex promoter segments. We found that compared with Taq RNAP, E. coli RNAP has much higher affinity only to the UP element and the downstream promoter duplex. This result indicates that the difference in stability between E. coli and Taq promoter complexes is mainly determined by the differential strength of core RNAP–DNA contacts. We suggest that the relative weakness of Taq RNAP interactions with DNA downstream of the transcription start point is the major reason of low stability and temperature sensitivity of promoter complexes formed by this enzyme.