Analysing the fitness cost of antibiotic resistance to identify targets for combination antimicrobials.

Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source of targets for combination antimicrobial therapies. Using transposon sequencing, we carried out a genome-wide analysis of fitness cost in a clinically common rpoB H526Y mutant. We find that genes whose products enable increased transcription elongation rates compound the fitness costs of resistance whereas genes whose products function in cell wall synthesis and division mitigate it. We validate our findings by showing that the cell wall synthesis and division defects of rpoB H526Y result from an increased transcription elongation rate that is further exacerbated by the activity of the uracil salvage pathway and unresponsiveness of the mutant RNAP to the alarmone ppGpp. We applied our findings to identify drugs that inhibit more readily rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic-resistant mutants should expedite the discovery of new combination therapies and delineate cellular pathways that underlie the molecular mechanisms of cost.

Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli.

Chemical modifications of RNA 5'-ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5'-end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5'-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded Ecoli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

A small molecule inhibits cell elongation by modulating cell wall polysaccharide composition in Arabidopsis.

The plant primary cell wall is comprised of pectin, cellulose and hemicelluloses, whose dynamic interactions play essential roles in plant cell elongation. Through a chemical genetics screening, we identified a small molecule, named cell wall modulator (CWM), which disrupted cell growth and deformed cell shape in etiolated Arabidopsis hypocotyl. A pectin defective mutant qua2, identified from screening an Arabidopsis EMS mutant library, showed a reduced sensitivity to CWM treatment. On the other hand, pectinase treatment suppressed the CWM induced phenotype. Furthermore, cellulose content was decreased in response to CWM treatment, while the cellulose synthesis mutants ixr1 and ixr2 were hypersensitive to CWM. Together, the study identified a small molecule CWM that induced a modification of the cell wall in elongating cells, likely through interfering with pectin modification. This molecule may be used as a tool to study cell wall remodeling during plant growth.

RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription.

All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the "σ-finger" of the bacterial transcription initiation factor σ. The structures reveal that-for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA-the "σ-finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.

Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn71, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.

Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor.

σS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNA polymerase (σS-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σS (σS2) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σS2 to promote the assembly of the σS-RNAP holoenzyme and also to facilitate formation of an RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

Structural basis for transcription antitermination at bacterial intrinsic terminator.

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.

Structures and mechanism of transcription initiation by bacterial ECF factors.

Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

Structural basis for transcription initiation by bacterial ECF σ factors.

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.

Identification of Auxin Activity Like 1, a chemical with weak functions in auxin signaling pathway.

KEY MESSAGE: A new synthetic auxin AAL1 with new structure was identified. Different from known auxins, it has weak effects. By AAL1, we found specific amino acids could restore the effects of auxin with similar structure. Auxin, one of the most important phytohormones, plays crucial roles in plant growth, development and environmental response. Although many critical regulators have been identified in auxin signaling pathway, some factors, especially those with weak fine-tuning roles, are still yet to be discovered. Through chemical genetic screenings, we identified a small molecule, Auxin Activity Like 1 (AAL1), which can effectively inhibit dark-grown Arabidopsis thaliana seedlings. Genetic screening identified AAL1 resistant mutants are also hyposensitive to indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). AAL1 resistant mutants such as shy2-3c and ecr1-2 are well characterized as mutants in auxin signaling pathway. Genetic studies showed that AAL1 functions through auxin receptor Transport Inhibitor Response1 (TIR1) and its functions depend on auxin influx and efflux carriers. Compared with known auxins, AAL1 exhibits relatively weak effects on plant growth, with 20 µM and 50 µM IC50 (half growth inhibition chemical concentration) in root and hypocotyl growth respectively. Interestingly, we found the inhibitory effects of AAL1 and IAA could be partially restored by tyrosine and tryptophan respectively, suggesting some amino acids can also affect auxin signaling pathway in a moderate manner. Taken together, our results demonstrate that AAL1 acts through auxin signaling pathway, and AAL1, as a weak auxin activity analog, provides us a tool to study weak genetic interactions in auxin pathway.

KDM4B as a target for prostate cancer: structural analysis and selective inhibition by a novel inhibitor.

The KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A-KDM4D), which selectively remove the methyl group(s) from tri/dimethylated lysine 9/36 of H3, modulate transcriptional activation and genome stability. The overexpression of KDM4A/KDM4B in prostate cancer and their association with androgen receptor suggest that KDM4A/KDM4B are potential progression factors for prostate cancer. Here, we report the crystal structure of the KDM4B·pyridine 2,4-dicarboxylic acid·H3K9me3 ternary complex, revealing the core active-site region and a selective K9/K36 site. A selective KDM4A/KDM4B inhibitor, 4, that occupies three subsites in the binding pocket is identified by virtual screening. Pharmacological and genetic inhibition of KDM4A/KDM4B significantly blocks the viability of cultured prostate cancer cells, which is accompanied by increased H3K9me3 staining and transcriptional silencing of growth-related genes. Significantly, a substantial portion of differentially expressed genes are AR-responsive, consistent with the roles of KDM4s as critical AR activators. Our results point to KDM4 as a useful therapeutic target and identify a new inhibitor scaffold.

Parallel screening of wild-type and drug-resistant targets for anti-resistance neuraminidase inhibitors.

Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA) drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR) influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT) and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye), which inhibited WT NA and MDR NA with IC(50) values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high mutation rates, such as cancer and human immunodeficiency virus type 1.

Core site-moiety maps reveal inhibitors and binding mechanisms of orthologous proteins by screening compound libraries.

Members of protein families often share conserved structural subsites for interaction with chemically similar moieties despite low sequence identity. We propose a core site-moiety map of multiple proteins (called CoreSiMMap) to discover inhibitors and mechanisms by profiling subsite-moiety interactions of immense screening compounds. The consensus anchor, the subsite-moiety interactions with statistical significance, of a CoreSiMMap can be regarded as a "hot spot" that represents the conserved binding environments involved in biological functions. Here, we derive the CoreSiMMap with six consensus anchors and identify six inhibitors (IC(50)<8.0 µM) of shikimate kinases (SKs) of Mycobacterium tuberculosis and Helicobacter pylori from the NCI database (236,962 compounds). Studies of site-directed mutagenesis and analogues reveal that these conserved interacting residues and moieties contribute to pocket-moiety interaction spots and biological functions. These results reveal that our multi-target screening strategy and the CoreSiMMap can increase the accuracy of screening in the identification of novel inhibitors and subsite-moiety environments for elucidating the binding mechanisms of targets.