The RimL transacetylase provides resistance to translation inhibitor microcin C.

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. 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.

RNA polymerase-promoter interactions determining different stability of the Escherichia coli and Thermus aquaticus transcription initiation complexes.

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.

A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.

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 ß 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.

Francisella RNA polymerase contains a heterodimer of non-identical α subunits.

BACKGROUND: All sequenced genomes of representatives of the Francisella genus contain two rpoA genes, which encode non-identical RNA polymerase (RNAP) subunits, α1 and α2. In all other bacteria studied to date, a dimer of identical α subunits initiates the assembly of the catalytically proficient RNAP core (subunit composition α2ßß'). Based on an observation that both α1 and α2 are incorporated into Francisella RNAP, Charity et al. (2007) previously suggested that up to four different species of RNAP core enzyme might form in the same Francisella cell. RESULTS: By in vitro assembly from fully denatured state, we determined that both Francisella α subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the α1 and α2 N-terminal domains (NTDs, responsible for dimerization). NTDs of α2 did not interact detectably, while weak interaction between α1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (ß', ß) and α1. No activity was observed with RNAP reconstitution reactions containing α2, while robust transcription activity was detected in reactions containing α1 and α2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within γ-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization. CONCLUSIONS: The results strongly suggest that most Francisella RNAP contains α heterodimer with a minor subfraction possibly containing α1 homodimer. Comparative sequence analysis suggests that this heterodimer is oriented, in a sense that only one monomer, α1, interacts with the ß subunit during the α2ß RNAP subassembly formation. Most likely the two rpoA copies in Francisella have emerged through a lineage-specific duplication followed by subfunctionalization of interacting paralogs.

The antibacterial threaded-lasso peptide capistruin inhibits bacterial RNA polymerase.

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 not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids 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 capistruin, 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, as a result of 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.