50 results found
Weinzierl ROJ, 2022, Robotic Affinity Purification of Soluble and Insoluble Recombinant Glutathione-S-Transferase Fusion Proteins., Methods Mol Biol, Vol: 2466, Pages: 93-109
A completely automated purification of glutathione-S-transferase (GST) fusion proteins, either in soluble form or after renaturation of insoluble inclusion bodies, is described. Depending on the expression levels and the amount of glutathione affinity matrix employed, the protocol yields approximately 30-100 μg of purified GST-fusion protein from 2 mL microplate cultures. The high yield is facilitated by employing an efficient chemical/enzymatic lysis procedure for preparing bacterial cell lysates. Insoluble GST-fusion proteins are automatically refolded by a high-throughput robotic microdialysis procedure that also assesses the degree of successful refolding by integrated GST enzymatic assays and quantitation of soluble protein successfully recovered after affinity purification. For soluble GST-fusion proteins the purification procedure is normally completed within 60 min, whereas urea-based denaturation-renaturation strategies typically require an additional 18 h. The integration of quantitation of cell growth and affinity-purified GST-fusion protein yield allows direct comparisons of different expression constructs and the yield of soluble GST-fusion proteins to be optimized in a systematic manner.
Weinzierl ROJ, 2021, Molecular dynamics simulations of human FOXO3 reveal intrinsically disordered regions spread spatially by intramolecular electrostatic repulsion, Biomolecules, Vol: 11, Pages: 1-16, ISSN: 2218-273X
The human transcription factor FOXO3 (a member of the ‘forkhead’ family of transcription factors) controls a variety of cellular functions that make it a highly relevant target for intervention in anti-cancer and anti-aging therapies. FOXO3 is a mostly intrinsically disordered protein (IDP). Absence of knowledge of its structural properties outside the DNA-binding domain constitutes a considerable obstacle to a better understanding of structure/function relationships. Here, I present extensive molecular dynamics (MD) simulation data based on implicit solvation models of the entire FOXO3/DNA complex, and accelerated MD simulations under explicit solvent conditions of a central region of particular structural interest (FOXO3120–530). A new graphical tool for studying and visualizing the structural diversity of IDPs, the Local Compaction Plot (LCP), is introduced. The simulations confirm the highly disordered nature of FOXO3 and distinguish various degrees of folding propensity. Unexpectedly, two ‘linker’ regions immediately adjacent to the DNA-binding domain are present in a highly extended conformation. This extended conformation is not due to their amino acid composition, but rather is caused by electrostatic repulsion of the domains connected by the linkers. FOXO3 is thus an IDP present in an unusually extended conformation to facilitate interaction with molecular interaction partners.
Nottebaum S, Weinzierl R, 2021, Transcribing genes the hard way: in vitro reconstitution of nanoarchaeal RNA polymerase reveals unusual active site properties, Frontiers in Molecular Biosciences, Vol: 8, ISSN: 2296-889X
Nanoarchaea represent a highly diverged archaeal phylum that displays many unusual biological features. The Nanoarchaeum equitans genome encodes a complete set of RNA polymerase (RNAP) subunits and basal factors. Several of the standard motifs in the active center contain radical substitutions that are normally expected to render the polymerase catalytically inactive. Here we show that, despite these unusual features, a RNAP reconstituted from recombinant Nanoarchaeum subunits is transcriptionally active. Using a sparse-matrix high-throughput screening method we identified an atypical stringent requirement for fluoride ions to maximize its activity under in vitro transcription conditions.
Genin N, Weinzierl R, 2020, Nucleotide loading modes of human RNA polymerase II as deciphered by molecular simulations, Biomolecules, Vol: 10, ISSN: 2218-273X
Mapping the route of nucleoside triphosphate (NTP) entry into the sequestered active site of RNA polymerase (RNAP) has major implications for elucidating the complete nucleotide addition cycle. Constituting a dichotomy that remains to be resolved, two alternatives, direct NTP delivery via the secondary channel (CH2) or selection to downstream sites in the main channel (CH1) prior to catalysis, have been proposed. In this study, accelerated molecular dynamics simulations of freely diffusing NTPs about RNAPII were applied to refine the CH2 model and uncover atomic details on the CH1 model that previously lacked a persuasive structural framework to illustrate its mechanism of action. Diffusion and binding of NTPs to downstream DNA, and the transfer of a preselected NTP to the active site, are simulated for the first time. All-atom simulations further support that CH1 loading is transcription factor IIF (TFIIF) dependent and impacts catalytic isomerization. Altogether, the alternative nucleotide loading systems may allow distinct transcriptional landscapes to be expressed.
Weinzierl R, Jeffery H, Weinzierl R, 2020, Multivalent and bidirectional binding of transcriptional transactivation domains to the MED25 coactivator, Biomolecules, Vol: 9, ISSN: 2218-273X
The human mediator subunit MED25 acts as a coactivator that binds the transcriptional activation domains (TADs) present in various cellular and viral gene-specific transcription factors. Previous studies, including on NMR measurements and site-directed mutagenesis, have only yielded low-resolution models that are difficult to refine further by experimental means. Here, we apply computational molecular dynamics simulations to study the interactions of two different TADs from the human transcription factor ETV5 (ERM) and Herpes virus VP16-H1 with MED25. Like other well-studied coactivator-TAD complexes, the interactions of these intrinsically disordered domains with the coactivator surface are temporary and highly dynamic (‘fuzzy’). Due to the fact that the MED25 TAD-binding region is organized as an elongated cleft, we specifically asked whether these TADs are capable of binding in either orientation and how this could be achieved structurally and energetically. Binding of both the ETV5 and VP16-TADs in either orientation appears to be possible but occurs in a conformationally distinct manner and utilizes different sets of hydrophobic residues present in the TADs to drive the interactions. We propose that MED25 and at least a subset of human TADs specifically evolved a redundant set of molecular interaction patterns to allow binding to particular coactivator binding without major prior spatial constraints.
Sullivan SS, Weinzierl ROJ, 2020, Optimization of Molecular Dynamics Simulations of c-MYC1-88-An Intrinsically Disordered System, LIFE-BASEL, Vol: 10
- Author Web Link
- Citations: 6
Erickson AFW, Deighan P, Garcia CP, et al., 2017, An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor, JOURNAL OF BACTERIOLOGY, Vol: 199, ISSN: 0021-9193
Scholes NS, Weinzierl ROJ, 2016, Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions, Plos Computational Biology, Vol: 12, ISSN: 1553-7358
Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of -helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between -helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators.
Wiesler SC, Weinzierl ROJ, 2015, Robotic High-Throughput Purification of Affinity-Tagged Recombinant Proteins, AFFINITY CHROMATOGRAPHY: METHODS AND PROTOCOLS, 3RD EDITION, Vol: 1286, Pages: 97-106, ISSN: 1064-3745
- Citations: 3
Nicod SS, Weinzierl RO, Burchell L, et al., 2014, Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA, Nucleic Acids Research, Vol: 42, Pages: 12523-12536, ISSN: 1362-4962
Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains. An emerging class of DNA-binding transcription factors, predominantly found in pathogenic bacteria interact with the DNA via a relatively novel type of DNA-binding domain, called the LytTR domain, which mainly comprises β strands. Even though the crystal structure of the LytTR domain of the virulence gene transcription factor AgrA from Staphylococcus aureus bound to its cognate DNA sequence is available, the contribution of specific amino acid residues in the LytTR domain of AgrA to transcription activation remains elusive. Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor. Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA.
Weinzierl ROJ, 2013, The RNA Polymerase Factory and Archaeal Transcription, CHEMICAL REVIEWS, Vol: 113, Pages: 8350-8376, ISSN: 0009-2665
- Author Web Link
- Citations: 12
Wiesler SC, Weinzierl RO, Buck M, 2013, An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity, Nucleic Acids Res, ISSN: 1362-4962
The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the sigma-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively 'RPi'). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in sigma54-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.
Wiesler SC, Werner F, Weinzierl RO, 2013, Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase, Methods Mol Biol, Vol: 977, Pages: 217-227, ISSN: 1940-6029
TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and record the formation of the first phosphodiester bonds. These assays can be used to measure the effect of transcription factors such as TFIIB and RNAP mutations on abortive transcription.
Wiesler SC, Weinzierl RO, 2012, High-throughput purification of affinity-tagged recombinant proteins, J Vis Exp, ISSN: 1940-087X
X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity. In order to attribute the different phenotypes to the nature of the mutation--rather than to fluctuating experimental conditions--it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates. Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex. We exploited the high-throughput purification method to genera
Heindl H, Greenwell P, Weingarten N, et al., 2011, Cation-pi interactions induce kinking of a molecular hinge in the RNA polymerase bridge helix domain, Biochem Soc Trans, Vol: 39, Pages: 31-35
Weinzierl ROJ, 2011, The Bridge Helix of RNA Polymerase Acts as a Central Nanomechanical Switchboard for Coordinating Catalysis and Substrate Movement, ARCHAEA-AN INTERNATIONAL MICROBIOLOGICAL JOURNAL, Vol: 2011, ISSN: 1472-3646
- Author Web Link
- Citations: 22
Weinzierl RO, Wiesler SC, 2011, Revealing the functions of TFIIB, Transcription, Vol: 2, Pages: 254-257, ISSN: 2154-1272
The TFIIB linker domain stimulates the catalytic activity of archaeal RNAP. By characterising a range of super-stimulating mutants we identified a novel rate-limiting step in transcription initiation. Our results help to interpret structural findings and pave the way towards higher-resolution structures of the RNAP-TFIIB linker interface.
Jovanovic M, Burrows PC, Bose D, et al., 2011, Activity map of the Escherichia coli RNA polymerase bridge helix, J Biol Chem, Vol: 286, Pages: 14469-14479, ISSN: 1083-351X
Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.
Weinzierl RO, 2010, The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain, BMC Biology, Vol: 8
Wiesler S, Weinzierl R, 2010, The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions, Nucleic Acids Res., Vol: 39, Pages: 464-474
Weinzierl ROJ, 2010, Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 38, Pages: 428-432, ISSN: 0300-5127
- Author Web Link
- Citations: 10
Camara B, Liu M, Reynolds J, et al., 2010, T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 107, Pages: 2247-2252, ISSN: 0027-8424
- Author Web Link
- Citations: 53
Tan L, Wiesler S, Trzaska D, et al., 2008, Bridge helix and trigger loop perturbations generate superactive RNA polymerases, J Biol, Vol: 7, ISSN: 1475-4924
BACKGROUND: Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. Two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. The functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal structures currently available. RESULTS: Using a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the Methanocaldococcus jannaschii RNA polymerase A' subunit, revealing a wide spectrum of in vitro phenotypes. We show that a surprisingly large number of single amino acid substitutions in the bridge helix, including a kink-inducing proline substitution, increase the specific activity of RNA polymerase. Other 'superactivating' substitutions are located in the adjacent base helices of the trigger loop. CONCLUSION: The results support the hypothesis that the nucleotide addition cycle involves a kinked bridge helix conformation. The active center of RNA polymerase seems to be constrained by a network of functional interactions between the bridge helix and trigger loop that controls fundamental parameters of RNA synthesis.
Nottebaum S, Tan L, Dominika T, et al., 2007, The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes., Nucleic Acids Research
Symposium BSGB, Roberts SGE, Weinzierl ROJ, et al., 2006, Transcription, Publisher: Portland Press, ISBN: 9781855781634
Werner F, Wiesler S, Nottebaum S, et al., 2006, Modulation of RNA polymerase core functions by basal transcription factor TFB/TFIIB, Biochem Soc Symp, Pages: 49-58, ISSN: 0067-8694
The archaeal basal transcriptional machinery consists of TBP (TATA-binding protein), TFB (transcription factor B; a homologue of eukaryotic TFIIB) and an RNA polymerase that is structurally very similar to eukaryotic RNA polymerase II. This constellation of factors is sufficient to assemble specifically on a TATA box-containing promoter and to initiate transcription at a specific start site. We have used this system to study the functional interaction between basal transcription factors and RNA polymerase, with special emphasis on the post-recruitment function of TFB. A bioinformatics analysis of the B-finger of archaeal TFB and eukaryotic TFIIB reveals that this structure undergoes rapid and apparently systematic evolution in archaeal and eukaryotic evolutionary domains. We provide a detailed analysis of these changes and discuss their possible functional implications.
Werner F, Weinzierl ROJ, 2005, Direct modulation of RNA polymerase core functions by basal transcription factors, MOLECULAR AND CELLULAR BIOLOGY, Vol: 25, Pages: 8344-8355, ISSN: 0270-7306
- Author Web Link
- Citations: 81
Ouhammouch M, Werner F, Weinzierl ROJ, et al., 2004, A fully recombinant system for activator-dependent archaeal transcription, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 279, Pages: 51719-51721, ISSN: 0021-9258
- Author Web Link
- Citations: 57
Werner F, Weinzierl RO, 2004, A recombinant archaeal RNA polymerase II-like model system, Recent Devel. Nucleic Acids Res., Vol: 1, Pages: 25-34
Werner F, Weinzierl ROJ, 2002, A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription, MOLECULAR CELL, Vol: 10, Pages: 635-646, ISSN: 1097-2765
- Author Web Link
- Citations: 114
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