Konstantin Kuznedelov

Structure-based analysis of RNA polymerase function: the largest subunit's rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA-DNA hybrid length

Analysis of multisubunit RNA polymerase (RNAP) structures revealed several elements that may constitute the enzyme‘s functional sites. One such element, the ‘rudder’, is formed by an evolutionarily conserved segment of the largest subunit of RNAP and contacts the nascent RNA at the upstream edge of the RNA–DNA hybrid, where the DNA template strand separates from the RNA transcript and re‐anneals with the non‐template strand. Thus, the rudder could (i) maintain the correct length of the RNA–DNA hybrid; (ii) stabilize the nascent RNA in the complex; and (iii) promote or maintain localized DNA melting at the upstream edge of the bubble. We generated a recombinant RNAP mutant that lacked the rudder and studied its properties in vitro. Our results demonstrate that the rudder is not required for establishment of the upstream boundary of the transcription bubble during promoter complex formation, nor is it required for separation of the nascent RNA from the DNA template strand or transcription termination. Our results suggest that the rudder makes critical contributions to elongation complex stability through direct interactions with the nascent RNA.

Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the alpha-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA(Asp) by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.

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.

A Consensus Adenine at Position –11 of the Nontemplate Strand of Bacterial Promoter Is Important for Nucleation of Promoter Melting

Numerous studies have suggested an important role of adenine at position –11 of the nontemplate strand of bacterial promoters for sequence-specific recognition of the –10 promoter element in single-stranded form. In this work, we attempted to identify a specific step in transcription initiation reaction that is most critically dependent on specific recognition of –11A. Mutating –11A in the context of a model promoter resulted in a profound decrease of the rate of heparin-resistant promoter complex formation and in a modest increase of the rate of heparin-resistant complex dissociation. The identity of nontemplate base at position –11 became relatively unimportant when the duplex in the vicinity of this position was destabilized by base pair mismatches. For promoters with a nonnative thymine at nontemplate position –11, we observed a remarkable correlation between the rate of heparin-resistant complex formation (or transcription activity) and the free energy of duplex stability in the vicinity of this residue, indicating that the replacement of –11A with a T affected a step in the reaction that involves local melting of DNA duplex. These data show that a promoter melting defect caused by a loss of RNA polymerase contact with –11A can be rescued by artificially induced local destabilization of the DNA duplex. These results are consistent with and support the idea that specific recognition of adenine at the nontemplate –11-position is important only for the initial nucleation of melting, which probably involves the flipping of this adenine out from the DNA duplex.

Chemical Synthesis Enables Biochemical and Antibacterial Evaluation of Streptolydigin Antibiotics

Inhibition of bacterial transcription represents an effective and clinically validated anti-infective chemotherapeutic strategy. We describe the evolution of our approach to the streptolydigin class of antibiotics that target bacterial RNA polymerases (RNAPs). This effort resulted in the synthesis and biological evaluation of streptolydigin, streptolydiginone, streptolic acid, and a series of new streptolydigin-based agents. Subsequent biochemical evaluation of RNAP inhibition demonstrated that the presence of both streptolic acid and tetramic acid subunits was required for activity of this class of antibiotics. In addition, we identified 10,11-dihydrostreptolydigin as a new RNAP-targeting agent, which was assembled with high synthetic efficiency of 15 steps in the longest linear sequence. Dihydrostreptolydigin inhibited three representative bacterial RNAPs and displayed in vitro antibacterial activity against S. salivarius . The overall increase in synthetic efficiency combined with substantial antibacterial activity of this fully synthetic antibiotic demonstrates the power of organic synthesis in enabling design and comprehensive in vitro pharmacological evaluation of new chemical agents that target bacterial transcription.

Interaction of T4 AsiA with its Target Sites in the RNA Polymerase σ70 Subunit Leads to Distinct and Opposite Effects on Transcription

Bacteriophage T4 AsiA is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of Escherichia coli RNA polymerase holoenzyme (Esigma(70)) and preventing promoter complex formation on most E.coli and early T4 promoters. In addition, Esigma(70)AsiA, but not Esigma(70), is a substrate of transcription activation by T4-encoded DNA-binding protein MotA, a co-activator of transcription from middle viral promoters. The molecular determinants of sigma(70)-AsiA interaction necessary for transcription inhibition reside in the sigma(70) conserved region 4.2, which recognizes the -35 promoter consensus element. The molecular determinants of sigma(70)-AsiA interaction necessary for MotA-dependent transcription activation have not been identified. Here, we show that in the absence of sigma(70) region 4.2, AsiA interacts with sigma(70) conserved region 4.1 and activates transcription in a MotA-independent manner. Further, we show that the AsiA dimer must dissociate to interact with either region 4.2 or region 4.1 of sigma(70). We propose that MotA may co-activate transcription by restricting AsiA binding to sigma(70) region 4.1.

Crystal Structure of Escherichia Coli Rnk, a New RNA Polymerase-Interacting Protein

Sequence-based searches identified a new family of genes in proteobacteria, named rnk, which shares high sequence similarity with the C-terminal domains of the Gre factors (GreA and GreB) and the Thermus/Deinococcus anti-Gre factor Gfh1. We solved the X-ray crystal structure of Escherichia coli regulator of nucleoside kinase (Rnk) at 1.9 A resolution using the anomalous signal from the native protein. The Rnk structure strikingly resembles those of E. coli GreA and GreB and Thermus Gfh1, all of which are RNA polymerase (RNAP) secondary channel effectors and have a C-terminal domain belonging to the FKBP fold. Rnk, however, has a much shorter N-terminal coiled coil. Rnk does not stimulate transcript cleavage in vitro, nor does it reduce the lifetime of the complex formed by RNAP on promoters. We show that Rnk competes with the Gre factors and DksA (another RNAP secondary channel effector) for binding to RNAP in vitro, and although we found that the concentration of Rnk in vivo was much lower than that of DksA, it was similar to that of GreB, consistent with a potential regulatory role for Rnk as an anti-Gre factor.

Analysis of the mechanism of nucleosome survival during transcription

Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones.

Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA

CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing.

Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions

Bacteriophages are bacterial viruses that infect bacterial cells, and they have developed ingenious mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step affinity isolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4-infected cells, we have identified bacteriophage T4-dependent modifications of the host RNA polymerase. We suggest that this methodology is broadly applicable for the identification of bacteriophage-dependent alterations of the host synthesis machinery.