Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, β'1 and β'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the β'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site.

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, β'1 and β'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the β'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site.

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including by sequence-specific interaction with a conserved pause-inducing TTNTTT motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP.

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization.

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σ factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units.

Comprehensive transcription terminator atlas for Bacillus subtilis.

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination.

Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism.

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M.

NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA.

NusA and NusG are transcription factors that stimulate RNA polymerase pausing in . While NusA was known to function as an intrinsic termination factor in , the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, Δ, and NusA depletion Δ strains.

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation.

Transcription initiation is a key checkpoint and highly regulated step of gene expression. The sigma (σ) subunit of RNA polymerase (RNAP) controls all transcription initiation steps, from recognition of the -10/-35 promoter elements, upon formation of the closed promoter complex (RPc), to stabilization of the open promoter complex (RPo) and stimulation of the primary steps in RNA synthesis. The canonical mechanism to regulate σ activity upon transcription initiation relies on activators that recognize specific DNA motifs and recruit RNAP to promoters.

Enhanced activity of Withania somnifera family-1 glycosyltransferase (UGT73A16) via mutagenesis.

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis.

RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase.

The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures.

Single-molecule analysis reveals the mechanism of transcription activation in .

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition.

Bacopa monniera recombinant mevalonate diphosphate decarboxylase: Biochemical characterization.

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.

Efficient shoots regeneration and genetic transformation of Bacopa monniera.

Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles.

Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala.

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions.

Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra.

Background: Gentiana scabra is commonly known as 'Longdan' is an important herb in traditional Chinese medicines, commonly used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are main bioactive compounds which attributed to the pharmacological properties of this plant. The use of hairy root cultures as an excellent alternative for the production of pharmaceutically important metabolites in less time period with ensured quality of raw materials.

Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.

Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb.

Molecular characterization and differential expression studies of an oxidosqualene cyclase (OSC) gene of Brahmi (Bacopa monniera).

Triterpenoid saponins are the class of secondary metabolites, synthesized via isoprenoid pathway. Oxidosqualene cyclases (OSCs) catalyzes the cyclization of 2, 3-oxidosqualene to various triterpene skeletons, the first committed step in triterpenoid biosynthesis. A full-length oxidosqualene cyclase cDNA from Bacopa monniera (BmOSC) was isolated and characterized.

Steady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants.

Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy.

Activation tagging in Salvia miltiorrhiza can cause increased leaf size and accumulation of tanshinone I and IIA in its roots.

Background: Salvia miltiorrhiza Bunge (Danshen), an important herb in traditional Chinese medicine, is commonly used for treatment of cardiovascular diseases. One of the major bioactive constituents of Danshen, diterpenoid tanshinone, has been proved with pharmacological properties and have the potential to be a new drug candidate against various diseases. In our previous study, we have established an activation tagging mutagenesis (ATM) population of callus lines of S.

Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles.

Functional characterization of a flavonoid glycosyltransferase gene from Withania somnifera (Ashwagandha).

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W.

Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1).

Unlabelled: : Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out.

Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies.

Unlabelled: Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.