Robotic Affinity Purification of Soluble and Insoluble Recombinant Glutathione-S-Transferase Fusion Proteins.

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.

Molecular Dynamics Simulations of Human FOXO3 Reveal Intrinsically Disordered Regions Spread Spatially by Intramolecular Electrostatic Repulsion.

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.

Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties.

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.

Nucleotide Loading Modes of Human RNA Polymerase II as Deciphered by Molecular Simulations.

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.

Multivalent and Bidirectional Binding of Transcriptional Transactivation Domains to the MED25 Coactivator.

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. The 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 coactivators without major prior spatial constraints.

Optimization of Molecular Dynamics Simulations of c-MYC1-88-An Intrinsically Disordered System.

Many of the proteins involved in key cellular regulatory events contain extensive intrinsically disordered regions that are not readily amenable to conventional structure/function dissection. The oncoprotein c-MYC plays a key role in controlling cell proliferation and apoptosis and more than 70% of the primary sequence is disordered. Computational approaches that shed light on the range of secondary and tertiary structural conformations therefore provide the only realistic chance to study such proteins. Here, we describe the results of several tests of force fields and water models employed in molecular dynamics simulations for the N-terminal 88 amino acids of c-MYC. Comparisons of the simulation data with experimental secondary structure assignments obtained by NMR establish a particular implicit solvation approach as highly congruent. The results provide insights into the structural dynamics of c-MYC1-88, which will be useful for guiding future experimental approaches. The protocols for trajectory analysis described here will be applicable for the analysis of a variety of computational simulations of intrinsically disordered proteins.

An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor.

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the ß' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σH, the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σH-directed gene expression but not in the activity of the stress-response sigma factor σB Pulldown experiments showed that the mutant RNAP was defective in associating with σH but could still associate with σA and σB The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the ß' coiled coil that is specifically detrimental for interaction with σH This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor.IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis ß' subunit whereby RNAPL257P associates with some σ factors (σA and σB) and enables vegetative cell growth but is defective in utilization of σH and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.

Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions.

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 α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators.

Robotic high-throughput purification of affinity-tagged recombinant proteins.

Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA.

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 ß 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.

An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity.

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 σ-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 σ(54)-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.

Promoter independent abortive transcription assays unravel functional interactions between TFIIB and RNA polymerase.

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.

High-throughput purification of affinity-tagged recombinant proteins.

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 generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase. Altogether, we generated, purified and analysed 381 mutants--a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results. This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

Activity map of the Escherichia coli RNA polymerase bridge helix.

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.

Cation-π interactions induce kinking of a molecular hinge in the RNA polymerase bridge-helix domain.

RNAPs (RNA polymerases) are complex molecular machines that contain a highly conserved catalytic site surrounded by conformationally flexible domains. High-throughput mutagenesis in the archaeal model system Methanocaldococcus jannaschii has demonstrated that the nanomechanical properties of one of these domains, the bridge-helix, exert a key regulatory role on the rate of the NAC (nucleotide-addition cycle). Mutations that increase the probability and/or half-life of kink formation in the BH-HC (bridge-helix C-terminal hinge) cause a substantial increase in specific activity ('superactivity'). Fully atomistic molecular dynamics simulations show that kinking of the BH-HC appears to be driven by cation-π interactions and involve amino acid side chains that are exceptionally highly conserved in all prokaryotic and eukaryotic species.

The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions.

RNA polymerases (RNAPs) require basal transcription factors to assist them during transcription initiation. One of these factors, TFIIB, combines promoter recognition, recruitment of RNAP, promoter melting, start site selection and various post-initiation functions. The ability of 381 site-directed mutants in the TFIIB 'linker domain' to stimulate abortive transcription was systematically quantitated using promoter-independent dinucleotide extension assays. The results revealed two distinct clusters (mjTFIIB E78-R80 and mjTFIIB R90-G94, respectively) that were particularly sensitive to substitutions. In contrast, a short sequence (mjTFIIB A81-K89) between these two clusters tolerated radical single amino acid substitutions; short deletions in that region even caused a marked increase in the ability of TFIIB to stimulate abortive transcription ('superstimulation'). The superstimulating activity did, however, not correlate with increased recruitment of the TFIIB/RNAP complex because substitutions in a particular residue (mjTFIIB K87) increased recruitment by more than 5-fold without affecting the rate of abortive transcript stimulation. Our work demonstrates that highly localized changes within the TFIIB linker have profound, yet surprisingly disconnected, effects on RNAP recruitment, TFIIB/RNAP complex stability and the rate of transcription initiation. The identification of superstimulating TFIIB variants reveals the existence of a previously unknown rate-limiting step acting on the earliest stages of gene expression.

The Bridge Helix of RNA polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement.

The availability of in vitro assembly systems to produce recombinant archaeal RNA polymerases (RNAPs) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying RNAP function. Over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. The Bridge Helix domain, which appears in numerous X-ray structures as a 35-amino-acid-long alpha helix, coordinates the concerted movement of several other domains during catalysis through kinking of two discrete molecular hinges. Mutations affecting these kinking mechanisms have a direct effect on the specific catalytic activity of RNAP and can in some instances more than double it. Molecular dynamics simulations have established themselves as exceptionally useful for providing additional insights and detailed models to explain the underlying structural motions.

Revealing the functions of TFIIB.

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.

The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain.

BACKGROUND: Cellular RNA polymerases (RNAPs) are complex molecular machines that combine catalysis with concerted conformational changes in the active center. Previous work showed that kinking of a hinge region near the C-terminus of the Bridge Helix (BH-H(C)) plays a critical role in controlling the catalytic rate. RESULTS: Here, new evidence for the existence of an additional hinge region in the amino-terminal portion of the Bridge Helix domain (BH-H(N)) is presented. The nanomechanical properties of BH-H(N) emerge as a direct consequence of the highly conserved primary amino acid sequence. Mutations that are predicted to influence its flexibility cause corresponding changes in the rate of the nucleotide addition cycle (NAC). BH-H(N) displays functional properties that are distinct from BH-H(C), suggesting that conformational changes in the Bridge Helix control the NAC via two independent mechanisms. CONCLUSIONS: The properties of two distinct molecular hinges in the Bridge Helix of RNAP determine the functional contribution of this domain to key stages of the NAC by coordinating conformational changes in surrounding domains.