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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.

In den Regelwerken zum passiven Schallschutz von Umfassungsbauteilen wird das erforderliche Schalldämm-Maß der Fassade als Einzahlwert entsprechend dem Bewertungsverfahren nach DIN EN ISO 717-1 [1] ermittelt. Um die spektrale Zusammensetzung verschiedener Lärmquellen und die frequenzabhängige Schalldämmung von Fassadenbauteilen zu berücksichtigen, werden in den einschlägigen Regelwerken Korrektursummanden bzw. Spektrum-Anpassungswerte verwendet. Im folgenden Beitrag wird der Einfluss verschiedener Außenlärmspektren und frequenz- abhängiger Schalldämm-Maße auf den Innenpegel diskutiert. Insbesondere werden die Unterschiede zwischen Holz- und Massivbauweise bezüglich des Schutzziels bzw. des Innenpegels betrachtet. Die Untersuchungen zeigen, dass keine generelle Differenzierung zwischen Leicht- und Massivbauweise erforderlich ist. Für hochschalldämmende Leichtbaukonstruktionen mit einem Ctr,50–5000 < –8 dB wird jedoch ein Korrekturterm für das erforderliche Fassaden-Schalldämm-Maß zur Sicherstellung des Schutzziels vorgeschlagen. Summary In the regulations for passive noise protection of surrounding components, the required sound reduction index of the facade is determined as a single value according to the assessment procedure according to DIN EN ISO 717-1 [1]. In order to take into account the spectral composition of different noise sources and the frequency-dependent sound insulation of facade components, correction summands or spectrum adaptation values are used in the relevant regulations. The following article discusses the influence of various outside noise spectra and frequency-dependent sound insulation measures on the inside level. In particular, the differences between wood and solid construction were considered with regard to the protection goal and the internal level. The investigations show that no general differentiation between lightweight and solid construction is necessary. For highly sound-insulating lightweight constructions with a Ctr, 50–5000 &nbsp

Die DIN 4109-2:2018 [2] sieht aufgrund der Frequenzzusammensetzung von Schienenverkehrsgeräuschen in Verbindung mit dem Frequenzspektrum der Schalldämm-Maße von Außenbauteilen eine pauschale Minderung des Beurteilungspegels von 5 dB vor. Inwieweit dieser Pauschalansatz, nachfolgend als Schienenkorrekturterm bezeichnet, ohne Differenzierung nach Zuggattungen und Fahrgeschwindigkeit Bestand haben wird, ist aktuell auch Thema im Schiedsverfahren zur DIN 4109 [2]. Die vorliegende Untersuchung, welche für eine Lochfassade mit geringem Fensterflächenanteil durchgeführt wurde, nimmt eine vergleichende Betrachtung der für innerstädtischen Schienenverkehrslärm und Straßenverkehrslärm ermittelten Korrektursummanden vor. Die Differenzen aus den Korrektursummanden für Schienenverkehr und Straßenverkehr werden mit dem bisherigen Ansatz zum Schienenkorrekturterm von 5 dB verglichen. Summary DIN 4109-2: 2018 [2] provides for a general reduction in the assessment level of 5 dB due to the frequency composition of rail traffic noise in connection with the frequency spectrum of the sound insulation dimensions of external components. The extent to which this flat-rate approach, hereinafter referred to as the rail correction term, will persist without differentiation according to the type of train and the speed of travel is currently also an issue in the arbitration proceedings to DIN 4109 [2]. The present investigation, which was carried out for a perforated facade with a small proportion of windows, takes a comparative look at the correction summaries determined for inner-city rail traffic noise and road traffic noise. The differences from the correction summands for rail traffic and road traffic are compared with the previous approach to the rail correction term of 5 dB.

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.

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.

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 (&lsquo, fuzzy&rsquo, ). 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.

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.

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 β′ subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σ H , 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 σ H -directed gene expression but not in the activity of the stress-response sigma factor σ B . Pulldown experiments showed that the mutant RNAP was defective in associating with σ H but could still associate with σ A and σ B . The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β′ coiled coil that is specifically detrimental for interaction with σ H . 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 β′ subunit whereby RNAP L257P associates with some σ factors (σ A and σ B ) and enables vegetative cell growth but is defective in utilization of σ H 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.

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.

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.

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 in-depth structure/function analysis of large protein complexes, such as RNA polymerases (RNAPs), requires an experimental platform capable of assembling variants of such enzymes in large numbers in a reproducible manner under defined in vitro conditions. Here we describe a streamlined and integrated protocol for assembling recombinant archaeal RNAPs in a high-throughput 96-well format. All aspects of the procedure including construction of redesigned expression plasmids, development of automated protein extraction/in vitro assembly methods and activity assays were specifically adapted for implementation on robotic platforms. The optimized strategy allows the parallel assembly and activity assay of 96 recombinant RNAPs (including wild-type and mutant variants) with little or no human intervention within 24 h. We demonstrate the high-throughput potential of this system by evaluating the side-chain requirements of a single amino acid position of the RNAP Bridge Helix using saturation mutagenesis.

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.

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.

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.

The archaeal and eukaryotic evolutionary domains diverged from each other approximately 2 billion years ago, but many of the core components of their transcriptional and translational machineries still display a readily recognizable degree of similarity in their primary structures. The F and P subunits present in archaeal RNA polymerases were only recently identified in a purified archaeal RNA polymerase preparation and, on the basis of localized sequence homologies, tentatively identified as archaeal versions of the eukaryotic RPB4 and RPB12 RNA polymerase subunits, respectively. We prepared recombinant versions of the F and P subunits from Methanococcus jannaschii and used them in in vitro and in vivo protein interaction assays to demonstrate that they interact with other archaeal subunits in a manner predicted from their eukaryotic counterparts. The overall structural conservation of the M. jannaschii F subunit, although not readily recognizable on the primary amino acid sequence level, is sufficiently high to allow the formation of an archaeal-human F-RPB7 hybrid complex.

Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each consisting of 12–18 different subunits. The evolutionarily highly conserved RNAP subunit RPB5 is shared by all three enzymes and therefore represents a key structural/functional component of all eukaryotic RNAPs. Here we present the crystal structure of the RPB5 subunit from Saccharomyces cerevisiae . The bipartite structure includes a eukaryote-specific N-terminal domain and a C-terminal domain resembling the archaeal RNAP subunit H. RPB5 has been implicated in direct protein-protein contacts with transcription factor IIB, one of the components of the RNAP II basal transcriptional machinery, and gene-specific activator proteins, such as the hepatitis B virus transactivator protein X. The experimentally mapped regions of RPB5 involved in these interactions correspond to distinct and surface-exposed α-helical structures.

RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

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.

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.

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from the in vitro assembly of the 12-subunit RNA polymerase from the hyperthermophile Methanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

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 1-5 . 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 6-8 . TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase 9-11 . 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 4,5,12 . 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 4 . 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.

Complex molecular machines are involved in all information-processing steps in cells. The handling of information-bearing macromolecules (predominantly nucleic acids and proteins) requires a range of catalytic capabilities, such as the sequence-specific synthesis of macromolecular entities from smaller precursors and further processing by breaking/joining of pre-existing molecular components. These catalytic activities have to be spatially and temporally precisely controlled to ensure accuracy and efficiency levels that are commensurate with the associated biological functions (Lyubimov et al., 2011). Unlike metabolic enzymes, which are often medium-sized enzymes with freely accessible active sites, the enzymes associated with replication, transcription, translation, recombination are typically large multi-subunit protein complexes capable of a complex conformational spectrum. This conformational spectrum manifests itself through the dynamic features of individual domains within these molecular machines: many of the domains act explicitly as nanomechanical elements by serving as allosteric sensors, molecular hinges and motor units (Bustamante et al., 2011; Heindl et al., 2011). A deeper understanding of the functions of molecular machines is currently a high-priority topic and ultimate success will strongly depend on a combination of ‘wet-lab’ experimental techniques (X-ray crystallography, NMR, spectroscopic methods, site-directed mutagenesis and chemical cross-linking) with sophisticated computational simulations. Fully atomistic molecular dynamics (MD) simulations offer the most comprehensive and detailed insights into the behavior of molecular entities and are therefore the method of choice for attempts to unravel the structural basis of molecular mechanisms (reviewed in Karplus & McCammon, 2002; Freddolino et al., 2010; McGeagh et al. 2011; Schlick et al., 2011).

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 TFIID complex consists of the TATA-binding protein (TBP) and associated factors (TAFs) serving to mediate transcriptional activation by promoter-specific regulators. Here we report the cloning of Drosophila TAFII250 and the assembly of a partial complex containing recombinant TBP, TAFII110 and the C-terminal domain of TAFII250. This triple complex supports Sp1 activation and reveals specific interactions between TAFII250, TBP and TAFII110.

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-HC) 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-HN) is presented. The nanomechanical properties of BH-HN 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-HN displays functional properties that are distinct from BH-HC, 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. Background RNA polymerases (RNAPs) play a central role in the regulation of gene expression. Like the majority of the enzymes involved in fundamental biological information-processing functions (for example, replication, transcription, recombination, repair), RNAPs are probably best viewed as intricate molecular machines. The movement of nucleic acid substrates, coupled with various types of active site chemistries, requires a precisely orchestrated sequence of conformational changes of protein domains during the transcription cycle (for recent reviews see [1-4]). The nanomechanical mechanisms guiding the structural rearrangements of domains within the active site are still very poorly understood. Thus far, models of the fundamental reaction catalyzed by RNAPs, the nucleotide addition cycle (NAC), have predominantly been derived from a series of crystal structures that contain RNAPs as apoenzymes (for example [5-9]), or complexed with various

RNAPs (RNA polymerases) are complex molecular machines containing structural domains that co-ordinate the movement of nucleic acid and nucleotide substrates through the catalytic site. X-ray images of bacterial, archaeal and eukaryotic RNAPs have provided a wealth of structural detail over the last decade, but many mechanistic features can only be derived indirectly from such structures. We have therefore implemented a robotic high-throughput structure–function experimental system based on the automatic generation and assaying of hundreds of site-directed mutants in the archaeal RNAP from Methanocaldococcus jannaschii. In the present paper, I focus on recent insights obtained from applying this experimental strategy to the bridge–helix domain. Our work demonstrates that the bridge–helix undergoes substantial conformational changes within a narrowly confined region (mjA′ Ala822-Gln823-Ser824) during the nucleotide-addition cycle. Naturally occurring radical sequence variations in plant RNAP IV and V enzymes map to this region. In addition, many mutations within this domain cause a substantial increase in the RNAP catalytic activity (‘superactivity’), suggesting that the RNAP active site is conformationally constrained.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the host RNA polymerase (RNAP)—a multi-subunit enzyme responsible for gene transcription—by a small (∼7 kDa) phage-encoded protein called Gp2. Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here we describe the first atomic resolution structure of Gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. Our comprehensive mutagenesis data reveal that two conserved arginine residues located on the opposite side of Gp2 are important for binding to and inhibition of RNAP. Based on a structural model of the Gp2-RNAP complex, we propose that inhibition of transcription by Gp2 involves prevention of RNAP-promoter DNA interactions required for stable DNA strand separation and maintenance of the “transcription bubble” near the transcription start site, an obligatory step in the formation of a transcriptionally competent promoter complex.

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.

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.