Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites

Biochemistry ◽  
1990 ◽  
Vol 29 (25) ◽  
pp. 5987-5994 ◽  
Author(s):  
Peter P. Chuknyisky ◽  
Joseph M. Rifkind ◽  
Edward Tarien ◽  
Richard B. Beal ◽  
Gunther L. Eichhorn
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Structural Studies ◽  
Rna Polymerase 1

Structural studies on the active site of Escherichia coli RNA polymerase. 2. Geometrical relationship of the interacting substrates

Biochemistry ◽  
1990 ◽  
Vol 29 (25) ◽  
pp. 5994-6002 ◽  
Author(s):  
Richard B. Beal ◽  
Rajasekharan P. Pillai ◽  
Peter P. Chuknyisky ◽  
Abraham Levy ◽  
Edward Tarien ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Structural Studies ◽  
Relationship Of ◽  
Geometrical Relationship

scholarly journals Hydroxo-Bridged Active Site of Flavodiiron NO Reductase Revealed by Spectroscopy and Computations

2020 ◽  
Author(s):  
Filipe Folgosa ◽  
Vladimir Pelmenschikov ◽  
Matthias Keck ◽  
Christian Lorent ◽  
Yoshitaka Yoda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Density Functional Theory ◽  
Data Collection ◽  
Active Site ◽  
Density Functional ◽  
Nuclear Resonance ◽  
Structural Studies ◽  
Functional Theory ◽  
Nuclear Resonance Vibrational Spectroscopy ◽  
Ligand Interactions

<p>NO and O<sub>2</sub> are detoxified in many organisms using flavodiiron proteins (FDPs). The exact coordination of the iron centre in the active site of these enzymes remains unclear despite numerous structural studies. Here, we used <sup>57</sup>Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand interactions in <i>Escherichia coli</i> FDP. This data combined with density functional theory (DFT) and <sup>57</sup>Fe Mössbauer spectroscopy indicate that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)–(µOH<sup>–</sup>)<sub>2</sub>–Fe(III) active site, while its reduction gives rise to a monohydroxo-diferrous Fe(II)–(µOH<sup>–</sup>)–Fe(II) site upon elimination of one bridging OH<sup>–</sup> ligand, thereby providing an open coordination site for NO binding. Prolonged NRVS data collection of the oxidised FDP resulted in photoreduction and formation of a partially reduced diiron center with two bridging hydroxyl ligands. These results have crucial implications for studying and understanding the mechanism of FDP as well as other non-haem diiron enzymes.</p>

scholarly journals Modulation of Monomer Conformation of the BglG Transcriptional Antiterminator from Escherichia coli

2004 ◽  
Vol 186 (20) ◽  
pp. 6775-6781 ◽  
Author(s):  
Liat Fux ◽  
Anat Nussbaum-Shochat ◽  
Livnat Lopian ◽  
Orna Amster-Choder
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Cross Linking ◽  
Structural Studies ◽  
Phosphorylated Proteins ◽  
Close Proximity ◽  
Compact Conformation

ABSTRACT The BglG protein positively regulates expression of the bgl operon in Escherichia coli by binding as a dimer to the bgl transcript and preventing premature termination of transcription in the presence of β-glucosides. BglG activity is negatively controlled by BglF, the β-glucoside phosphotransferase, which reversibly phosphorylates BglG according to β-glucoside availability, thus modulating its dimeric state. BglG consists of an RNA-binding domain and two homologous domains, PRD1 and PRD2. Based on structural studies of a BglG homologue, the two PRDs fold similarly, and the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have recently shown that the affinity between PRD1 and PRD2 of BglG is high, and a fraction of the BglG monomers folds in the cell into a compact conformation, in which PRD1 and PRD2 are in close proximity. We show here that both BglG forms, the compact and noncompact, bind to the active site-containing domain of BglF, IIBbgl, in vitro. The interaction of BglG with IIBbgl or BglF is mediated by PRD2. Both BglG forms are detected as phosphorylated proteins after in vitro phosphorylation with IIBbgl and are dephosphorylated by BglF in vitro in the presence of β-glucosides. Nevertheless, genetic evidence indicates that the interaction of IIBbgl and BglF with the compact form is seemingly less favorable. Using in vivo cross-linking, we show that BglF enhances folding of BglG into a compact conformation, whereas the addition of β-glucosides reduces the amount of this form. Based on these results we suggest a model for the modulation of BglG conformation and activity by BglF.

scholarly journals Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 87
Author(s):  
Caleb R. Schlachter ◽  
Andrea O’Malley ◽  
Linda L. Grimes ◽  
John J. Tomashek ◽  
Maksymilian Chruszcz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Drugs Of Abuse ◽  
Biochemical Characterization ◽  
Genomic Data ◽  
Organic Substrates ◽  
Structural Studies ◽  
Biotechnological Applications ◽  
Sulfatase Activity

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

scholarly journals DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

1999 ◽  
Vol 63 (2) ◽  
pp. 457-478 ◽  
Author(s):  
Benoit Coulombe ◽  
Zachary F. Burton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Dna Bending ◽  
Tata Binding Protein ◽  
Transcriptional Activators ◽  
Catalytic Center ◽  
Transcription Bubble ◽  
Rna Polymerase Ii Transcription

SUMMARY A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription.

Artificial Evolution of an Enzyme Active Site: Structural Studies of Three Highly Active Mutants of Escherichia coli Alkaline Phosphatase

2002 ◽  
Vol 316 (4) ◽  
pp. 941-953 ◽  
Author(s):  
M-H.Le Du ◽  
C. Lamoure ◽  
B.H. Muller ◽  
O.V. Bulgakov ◽  
E. Lajeunesse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Active Site ◽  
Artificial Evolution ◽  
Structural Studies ◽  
Enzyme Active Site ◽  
Highly Active

scholarly journals DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

2015 ◽  
Vol 112 (50) ◽  
pp. E6862-E6871 ◽  
Author(s):  
Andrey Parshin ◽  
Anthony L. Shiver ◽  
Jookyung Lee ◽  
Maria Ozerova ◽  
Dina Schneidman-Duhovny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Amino Acid Starvation ◽  
Cross Linking ◽  
Molecular Function ◽  
Secondary Channel ◽  
Global Response ◽  
Microbial Life ◽  
Active Site Region

Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo–cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.

scholarly journals Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jin Young Kang ◽  
Paul Dominic B Olinares ◽  
James Chen ◽  
Elizabeth A Campbell ◽  
Arkady Mustaev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Terminal Segment ◽  
Structural Basis ◽  
Elongation Complex ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Active Site Cleft

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β’ subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.

Structural Studies of Escherichia coli RNA Polymerase

1998 ◽  
Vol 63 (0) ◽  
pp. 269-276 ◽  
Author(s):  
S.A. DARST ◽  
A. POLYAKOV ◽  
C. RICHTER ◽  
G. ZHANG
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Structural Studies
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