SummaryTo address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.
Condensing enzymes are essential in type II fatty acid synthesis and are promising targets for antibacterial drug discovery. Recently, a new approach using a xylose-inducible plasmid to express antisense RNA in Staphylococcus aureus has been described; however, the actual mechanism was not delineated. In this paper, the mechanism of decreased target protein production by expression of antisense RNA was investigated using Northern blotting. This revealed that the antisense RNA acts posttranscriptionally by targeting mRNA, leading to 5 mRNA degradation. Using this technology, a two-plate assay was developed in order to identify FabF/ FabH target-specific cell-permeable inhibitors by screening of natural product extracts. Over 250,000 natural product fermentation broths were screened and then confirmed in biochemical assays, yielding a hit rate of 0.1%. All known natural product FabH and FabF inhibitors, including cerulenin, thiolactomycin, thiotetromycin, and Tü3010, were discovered using this whole-cell mechanism-based screening approach. Phomallenic acids, which are new inhibitors of FabF, were also discovered. These new inhibitors exhibited target selectivity in the gel elongation assay and in the whole-cell-based two-plate assay. Phomallenic acid C showed good antibacterial activity, about 20-fold better than that of thiolactomycin and cerulenin, against S. aureus. It exhibited a spectrum of antibacterial activity against clinically important pathogens including methicillinresistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae.Hundreds of essential proteins have been identified in bacteria as potential drug targets (1,16,18,23). Of these, only a few are targets of therapeutically useful drugs. These include penicillin binding proteins, D-Ala-D-Ala ligase, MurA, undecaprenyl pyrophosphate, and alanine racemase for cell wall; 30S and 50S ribosomal subunits, elongation factor G, and IletRNA synthetase for protein synthesis; RNA polymerase for RNA synthesis; InhA (FabI) for fatty acid synthesis; dihydrofolate reductase (FolA) and p-aminobenzoic acid synthase (FolP) for metabolism; and DNA gyrase and topoisomerase IV for DNA synthesis. In past decades, extensive chemical modification of existing antibiotics has afforded improved activity against their targets. This strategy served well to develop new and effective antibiotics; however, such modification is becoming increasingly difficult and identification of new classes of compounds with different modes of action is critical to combat emerging resistance and meet clinical needs.
Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a highly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000, but which did not contain the TATA-binding protein (TBP). This complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rm6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.
RRN3 is one of the RRN genes specifically required for the transcription of rDNA by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. We have cloned the gene, determined the nucleotide sequence, and found that it is an essential gene which encodes a protein of calculated molecular weight of 72 369. Extracts prepared from rrn3 mutants were defective in in vitro transcription of rDNA templates. We used extracts from a strain containing an epitope‐tagged Rrn3 protein to purify a factor that could complement the mutant extracts. Using immunoaffinity purification combined with Mono Q chromatography, we obtained an essentially pure preparation of Rrn3p which complements the mutant extracts. By carrying out template commitment experiments, we found that Rrn3p is not part of the pre‐initiation complex that is stable through multiple rounds of transcription. We also found that pre‐incubation of Rrn3p with purified Pol I leads to stimulation of transcription upon subsequent mixing with DNA template and other transcription reaction components. Single‐round transcription experiments using the detergent Sarkosyl showed that this stimulation is due to increased efficiency of formation of a Sarkosyl‐resistant pre‐initiation complex. Thus, Rrn3p appears to interact directly with Pol I, apparently stimulating Pol I recruitment to the promoter, and is distinct from two other Pol I‐specific transcription factors, the Rrn6/7 complex and the Rrn5/9/10 complex (UAF), characterized previously.
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