Synthesis of streptolydigin, a potent bacterial RNA polymerase inhibitor

Streptolydigin is a highly potent, broad-spectrum antibiotic produced by Streptomyces lydicus, which inhibits bacterial RNA polymerase. We describe the first synthesis of streptolydigin, which was assembled in a highly convergent and fully stereocontrolled fashion with a longest linear sequence of 24 steps starting from commercially available precursors. The assembly process entailed preparation of fully elaborated streptolic and ydiginic subunits of the natural product, followed by a highly efficient union in a three-step one-pot procedure, which included Dieckmann cyclization with a concomitant imide opening, Horner-Wadsworth-Emmons olefination, and desilylation.     

Characterization of the active site of yeast RNA polymerase II by DFT and ReaxFF calculations

Most known DNA-dependent RNA polymerases (RNAPs) share a universal heptapeptide, called the NADFDGD motif. The crystal structures of RNAPs indicate that in all cases this motif forms a loop with an embedded triad of aspartic acid residues. This conserved loop is the key part of the active site. Based on the crystal structures of the yeast RNAP II, we have studied this common active site for three cases: (1) single RNAP, (2) pre-translocation elongation complex, and (3) post-translocation elongation complex. Here we have applied two different modeling methods, the GGA density functional theory method (PBE) of quantum mechanics (QM) and the ReaxFF reactive force field. The QM calculations indicate that the loop shrinks from pre- to post-translocation and expands from post- to pre- translocation. In addition, PBE MD simulations in the gas phase at 310 K shows that the loop in the single-RNAP case is tightly connected to a catalytic Mg ²⁺ ion and that there is an ordered hydrogen bond network in the loop. The corresponding ReaxFF MD simulation presents a less stable loop structure, suggesting that ReaxFF may underestimate the coordinating interactions between carbonyl oxygen and magnesium ion compared to the gas phase QM. However, with ReaxFF it was practical to study the dynamics for a much more detailed model for the post-translocational case, including the complete loop and solvent. This leads to a plausible reactant-side model that may explain the large difference in efficiency of NTP polymerization between RNA and DNA polymerases.     

A theoretical study of the mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II

The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II has been investigated using molecular mechanics and quantum mechanics methods.Molecular dynamics(MD) simulations were carried out using the TIP3 water model and generalized solvent boundary potential(GSBP) by CHARMM based on the X-ray crystal structure.Two models of the ternary elongation complex were constructed based on CHARMM MD calculations.All the species including reactants,transition states,intermediates,and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2.Three pathways were explored using the DFT method.The most favorable reaction pathway involves indirect proton migration from the RNA primer 3’-OH to the oxygen atom of-phosphate via a solvent water molecule,proton rotation from the oxygen atom of-phosphate to the-phosphate side,the RNA primer 3’-O nucleophilic attack on the-phosphorus atom,and P-O bond breakage.The corresponding reaction potential profile was obtained.The rate limiting step,with a barrier height of 21.5 kcal/mol,is the RNA primer 3’-O nucleophilic attack,rather than the commonly considered proton transfer process.A high-resolution crystal structure including crystallographic water molecules is required for further studies.     

Structural and functional analysis of T7D promoter and its complex withE. coli RNA polymerase

The DNA fragment containing D promoter of the T7 bacteriophage and its complex with DNA-dependent RNA polymerase undergo a conformational transition at 30 °C, which is accompanied by an increase in the rate of phosphodiester bond synthesis.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1339–1374, July, 1995.The present work was supported by the International Science Foundation (project No. RMY300) and the Russian Foundation for Basic Research (project No. 94-04-11306a).     

Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail

Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.     

Convenient synthesis of nucleoside 5'-triphosphates for RNA transcription

By generating a selective phosphitylating reagent in situ, nucleoside 5'-triphosphates can be conveniently synthesized in one pot. This novel strategy without nucleoside protection has been developed to largely simplify synthesis of the nucleoside triphosphates. This demonstrated principle can be applied to the 5'-triphosphate synthesis of both native and modified nucleosides.     

Nanolitre droplet array for real time reverse transcription polymerase chain reaction

Recently, more and more effort has been put into the miniaturization of genetic tests such as quantitative PCR (qPCR), because it is no doubt a powerful tool for molecular diagnosis and quantitative biology. In this paper, we developed a low density nanolitre droplet array generated on a chemical modified silicon chip for gene quantification. Reliable and sensitive two step real time qRT-PCR assay for microRNA measurement was performed within 500 nL droplets. It has a dynamic range of six orders of magnitude, allowing for the quantification of microRNA input from 10(3) to 10(9) copies per reaction. We successfully applied the platform for quantitative measurement of mir-122 across five cultured cell lines. The minimum total RNA input was as low as 1 pg per assay, which showed great potential for gene quantification at single cell level. We envision the droplet based qPCR chip would be a universal and low-cost platform for gene quantification in ordinary biological laboratories.