Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

The general transcription factor TFIID is composed of the TATA-box-binding protein (TBP) and a set of TBP-associated factors (TAFIIs). In vitro, TAFIIs are required for activated transcription, and have been proposed to be obligatory targets of transcriptional activator proteins (activators)2. The function of TAFIIs has not been investigated systematically in vivo. A Saccharomyces cerevisiae TAFII complex (yTAFII complex) has been identified that shares functional and structural similarities with higher eukaryotic TFIID. In particular, most yTAFIIs are the homologue of a higher eukaryotic TAFII. Here we report that inactivation or depletion of six different yTAFIIs, including the core yTAFII, that contacts TBP, does not compromise transcriptional activation. We conclude that in vivo, activated transcription of many genes can occur in the absence of functional yTAFIIS, and that in these instances another transcription component(s) must be the target of the activator.

In higher eukaryotes the RNA polymerase II transcription factor TFIID is composed of a TATA-box-binding protein (TBP) and a set of tightly bound polypeptides, designated TBP-associated factors (TAFIIS). One or more TAFIIS are coactivators that are required for activated but not basal transcription. The eukaryotic transcription machinery is highly conserved and it is therefore puzzling that TAFIIS have not been identified in yeast. Here we use TBP as a protein-affinity ligand to isolate from yeast a multisubunit complex that is required specifically for activated transcription by RNA polymerase II. Microsequence analysis and cloning of two subunits of this complex reveal that they are the homologues of known mammalian and Drosophila TAFIIS. The genes encoding these two yeast TAFIIS are essential, suggesting that activated transcription is required for viability of Saccharomyces cerevisiae.

To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10 8 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [ 14 C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14␣-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

TFIID comprises the TATA box-binding protein and a set of highly conserved associated factors (TAF(II)s). yTAF(II)145, the core subunit of the yeast TAF(II) complex, is dispensable for transcription of most yeast genes but specifically required for progression through G1/S. Here we show that transcription of G1 and certain B-type cyclin genes is dependent upon yTAF(II)145. At high cell density or following nutrient deprivation, yeast cells cease division, enter a G0-like state, and terminate transcription of most genes. In this stationary phase, we find that the levels of yTAF(II)145, several other yTAF(II)s, and TBP are drastically reduced. Collectively, our results indicate that yTAF(II)145 and other TFIID components have a specialized role in transcriptional regulation of cell cycle progression and growth control.