Open Promoter Complex Formation Research Articles

A saponin-polybromophenol antibiotic (CU1) from Cassia fistula Bark Against Multi-Drug Resistant Bacteria Targeting RNA polymerase

BackgroundGradual increase of multidrug resistant infections is a threat to the human race as MDR plasmids have acquired.>10 mdr and drug efflux genes to inactivate antibiotics. Plants secret anti-metabolites to retard growth of soil and water bacteria and are ideal source of antibiotics. PurposePurpose of the study is to discover an alternate phyto-drug from medicinal plants of India that selectively kills MDR bacteria. MethodsMDR bacteria isolated from Ganga river water, milk, chicken meat and human hair for testing phyto-extracts. Eighty medicinal plants were searched and six phyto-extracts were selected having good antibacterial activities as demonstrated by agar-hole assays giving 15 ​mm or greater lysis zone. Phyto-extracts were made in ethanol or methanol (1:5 w/v) for overnight and were concentrated. Preparative TLC and HPLC were performed to purify phytochemical. MASS, NMR, FTIR methods were used for chemical analysis of CU1. In vitro RNA polymerase and DNA polymerase assays were performed for target identification. ResultsCU1 belongs to a saponin bromo-polyphenol compound with a large structure that purified on HPLC C18 column at 3min. CU1 is bacteriocidal but three times less active than rifampicin in Agar-hole assay. While in LB medium it shows greater than fifteen times poor inhibitor due to solubility problem. CU1 inhibited transcription from Escherichia coli as well as Mycobacterium tuberculosis RNA Polymerases. Gel shift assays demonstrated that CU1 interferes at the open promoter complex formation step. On the other hand CU1 did not inhibit DNA polymerase. ConclusionPhyto-chemicals from Cassia fistula bark are abundant, less toxic, target specific and may be a safer low cost drug against MDR bacterial diseases.

Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli.

Modulating resource allocation in bacteria to redirect metabolic building blocks to the formation of recombinant proteins rather than biomass formation remains a grand challenge in biotechnology. Here, we present a novel approach for improved recombinant protein production (RPP) using Escherichia coli (E. coli) by decoupling recombinant protein synthesis from cell growth. We show that cell division and host mRNA transcription can be successfully inhibited by coexpression of a bacteriophage-derived E. coli RNA polymerase (RNAP) inhibitor peptide and that genes overtranscribed by the orthogonal T7 RNAP can finally account to >55% of cell dry mass (CDM). This RNAP inhibitor peptide binds the E. coli RNAP and therefore prevents σ-factor 70 mediated formation of transcriptional qualified open promoter complexes. Thereby, the transcription of σ-factor 70 driven host genes is inhibited, and metabolic resources can be exclusively utilized for synthesis of the protein of interest (POI). Here, we mimic the late phase of bacteriophage infection by coexpressing a phage-derived xenogeneic regulator that reprograms the host cell and thereby are able to significantly improve RPP under industrial relevant fed-batch process conditions at bioreactor scale. We have evaluated production of several different recombinant proteins at different scales (from microscale to 20 L fed-batch scale) and have been able to improve total and soluble proteins yields up to 3.4-fold in comparison to the reference expression system E. coli BL21(DE3). This novel approach for growth-decoupled RPP has profound implications for biotechnology and bioengineering and helps to establish more cost-effective and generic manufacturing processes for biologics and biomaterials.

The role of yeast RNA polymerase I initiation factor Core Factor subunit Rrn7 in promoter open complex formation

A central component in S. cerevisiae RNA polymerase I (Pol I) pre‐initiation complex (PIC) formation and transcriptional regulation is the evolutionarily conserved initiation factor termed Core Factor (CF). CF is a heterotrimer composed of Rrn6, Rrn11, and the TFIIB‐related factor Rrn7. Recent Cryo‐EM structures of the Pol I initiation complex reveal that CF resembles a right hand grasping promoter DNA in which key contacts are mediated by Rrn11 and Rrn7 (Han et al., eLife, 2017). Similar to its Pol II counterpart TFIIB, CF subunit Rrn7 consists of an N‐terminal zinc ribbon (ZR) domain followed by a linker region and two cyclin fold repeats. We systematically mutagenized the Rrn7 ZR and linker domains to further understand their role in Pol I transcription initiation. We found that ZR and linker mutants are still able to form the CF complex and assemble a PIC, yet are defective in promoter opening. These findings, paired with the Cryo‐EM structure, suggest that CF acts as a ratchet to drive promoter DNA into the active site of Pol I and that the Rrn7 ZR and linker domains play a dynamic role in ATP‐independent promoter opening.Support or Funding InformationThis work was supported by grants awarded to BAK: National Cancer Institute (5K22CA184235), Alexandrine and Alexander L. Sinsheimer Scholar award, and SUNY Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Thermus phage protein inhibits host RNA polymerase by preventing template DNA strand loading during open promoter complex formation.

RNA polymerase (RNAP) is a major target of gene regulation. Thermus thermophilus bacteriophage P23–45 encodes two RNAP binding proteins, gp39 and gp76, which shut off host gene transcription while allowing orderly transcription of phage genes. We previously reported the structure of the T. thermophilus RNAP•σA holoenzyme complexed with gp39. Here, we solved the structure of the RNAP•σA holoenzyme bound with both gp39 and gp76, which revealed an unprecedented inhibition mechanism by gp76. The acidic protein gp76 binds within the RNAP cleft and occupies the path of the template DNA strand at positions –11 to –4, relative to the transcription start site at +1. Thus, gp76 obstructs the formation of an open promoter complex and prevents transcription by T. thermophilus RNAP from most host promoters. gp76 is less inhibitory for phage transcription, as tighter RNAP interaction with the phage promoters allows the template DNA to compete with gp76 for the common binding site. gp76 also inhibits Escherichia coli RNAP highlighting the template–DNA binding site as a new target site for developing antibacterial agents.

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies.

Subunit Dynamics and Nucleotide-Dependent Asymmetry of an AAA+ Transcription Complex

Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA+ protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA+ domain of one well-studied bEBP in complex with its substrate, the σ54 subunit of RNA polymerase. Our results demonstrate that the free AAA+ domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ54 binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs.

Structure and function of CarD, an essential mycobacterial transcription factor

CarD, an essential transcription regulator in Mycobacterium tuberculosis, directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates.

The X‐ray Crystal Structure of Escherichia Coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the best‐studied bacterial RNAP and has been used as the model RNAP for potential RNAP‐targeting antibiotic screening and evaluation. However, the X‐ray crystal structure of E. coli RNAP has been limited to individual domains. In this study, E. coli RNAP core enzyme (E) and holoenzyme (Eσ70) were prepared from overexpressed proteins. The holoenzyme was crystallized and solved its X‐ray structure at 3.8 Å resolution. The E. coli holoenzyme structure shows the σ region 1.1 (σ1.1) and α subunit C‐terminal domain (αCTD) structures for first time in any bacterial RNAP. The structural comparison between the E. coli and Thermus RNAPs shows that the ω subunit structure is different in E. coli from that in Thermus that may be related to the fact that the only E. coli RNAP is able to respond ppGpp for transcription regulation. The σ1.1 is positioned at the RNAP DNA binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that the σ1.1 contains a basic patch on its surface, which may play an important role in the DNA interaction to facilitate the transition from the closed to open promoter complexes. The αCTD is positioned next to the σ domain 4 (σ4) with a fully stretched linker between the N‐ and C‐terminal domains. E. coli RNAP crystals can be conveniently prepared from overexpressed proteins, allowing further structural studies of bacterial RNAP mutants, including antibiotic resistant RNAPs.

X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ(70) holoenzyme, which shows σ region 1.1 (σ1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.

RNA polymerase III subunit architecture and implications for open promoter complex formation

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine cross-linking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization.

Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised

The Escherichia coli-infecting bacteriophage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 phage development. The interaction site for Gp2 on the E. coli RNAp is the β′ jaw domain, which is part of the DNA binding channel. The binding of Gp2 to the β′ jaw antagonizes several steps associated with interactions between the RNAp and promoter DNA, leading to inhibition of transcription at the open promoter complex formation step. In the structure of the complex formed between Gp2 and a fragment of the β′ jaw, amino acid residues in the β3 strand of Gp2 contribute to the primary interaction interface with the β′ jaw. The 7009 E. coli strain is resistant to T7 because it carries a charge reversal point mutation in the β′ jaw that prevents Gp2 binding. However, a T7 phage encoding a mutant form of Gp2, called Gp2β, which carries triple amino acid substitutions E24K, F27Y and R56C, can productively infect this strain. By studying the molecular basis of inhibition of RNAp from the 7009 strain by Gp2β, we provide several lines of evidence that the E24K and F27Y substitutions facilitate an interaction with RNAp when the primary interaction interface with the β′ jaw is compromised. The proposed additional interaction interface between RNAp and Gp2 may contribute to the multipronged mechanism of transcription inhibition by Gp2.

A dual switch controls bacterial enhancer-dependent transcription.

Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation.

Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.

Formation of MgF3−-dependent complexes between an AAA+ ATPase and σ54

The widely distributed bacterial σ54-dependent transcription regulates pathogenicity and numerous adaptive responses in diverse bacteria. Formation of the σ54-dependent open promoter complex is a multi-step process driven by AAA+ ATPases. Non-hydrolysable nucleotide analogues are particularly suitable for studying such complexity by capturing various intermediate states along the energy coupling pathway. Here we report a novel ATP analogue, ADP–MgF3−, which traps an AAA+ ATPase with its target σ54. The MgF3−-dependent complex is highly homogeneous and functional assays suggest it may represent an early transcription intermediate state valuable for structural studies.

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP–gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the −10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.

A new basal promoter element recognized by RNA polymerase core enzyme

Bacterial promoters are recognized by RNA polymerase (RNAP) σ subunit, which specifically interacts with the -10 and -35 promoter elements. Here, we provide evidence that the β' zipper, an evolutionarily conserved loop of the largest subunit of RNAP core, interacts with promoter spacer, a DNA segment that separates the -10 and -35 promoter elements, and facilitates the formation of stable closed promoter complex. Depending on the spacer sequence, the proposed interaction of the β' zipper with the spacer can also facilitate open promoter complex formation and even substitute for interactions of the σ subunit with the -35 element. These results suggest that there exists a novel class of promoters that rely on interaction of the β' zipper with promoter spacer, along with or instead of interactions of σ subunit with the -35 element, for their activity. Finally, our data suggest that sequence-dependent interactions of the β' zipper with DNA can contribute to promoter-proximal σ-dependent RNAP pausing, a recently recognized important step of transcription control.

Molecular Insights into the Control of Transcription Initiation at the Staphylococcus aureus agr operon

The accessory gene regulatory (agr) operon of the opportunistic human pathogen Staphylococcus aureus is a prime pathogenesis factor in this bacterium. The agr operon consists of two transcription units, RNAII and RNAIII, which are transcribed from divergent promoters, P2 and P3, respectively. RNAII encodes a quorum-sensing system, including AgrA, the master transcription activator of the agr operon. RNAIII is the effector RNA molecule that regulates the expression of many virulence genes. Owing to the atypical spacer lengths of P2 and P3, it is widely considered that transcription from P2 and P3 only occurs in a strictly AgrA-dependent manner. Here, using a fully native S. aureus in vitro transcription system, we provide the first molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, the results demonstrate that RNA polymerase (RNAp) can interact with P2 and P3 equally well in the absence of AgrA. However, formation of a transcription-competent open promoter complex (RPo) occurs more readily at P2 than at P3 when AgrA is absent. Reducing the atypical P3 spacer region length to the optimal length of 17 nucleotides significantly improves promoter activity by facilitating the isomerisation of the initial RNAp–P3 complexes to RPo, and the extended − 10-like element of P3 is required for optimal promoter activity. AgrA increases the occupancy of both promoters by RNAp and thereby increases the amount of transcription initiated at P2 and P3. However, the AgrA-mediated effect on transcription initiation is more prominent at P3 that at P2. The effect of AgrA at P2 and P3 appears to be restricted to events leading to the formation of RPo. The relevance of AgrA-independent and AgrA-dependent transcription initiation at P2 and P3 is presented in the context of our current understanding of the role of the agr operon in the pathobiology of S. aureus.

A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

Nucleation of promoter melting in bacteria is coupled with RNA polymerase (RNAP) binding to a conserved -10 promoter element located at the upstream edge of the transcription bubble. The mechanism of downstream propagation of the transcription bubble to include the transcription start site is unclear. Here we introduce new model downstream fork junction promoter fragments that specifically bind RNAP and mimic the downstream segment of promoter complexes. We demonstrate that RNAP binding to downstream fork junctions is coupled with DNA melting around the transcription start point. Consequently, certain downstream fork junction probes can serve as transcription templates. Using a protein beacon fluorescent method, we identify structural determinants of affinity and transcription activity of RNAP-downstream fork junction complexes. Measurements of RNAP interaction with double-stranded promoter fragments reveal that the strength of RNAP interactions with downstream DNA plays a critical role in promoter opening and that the length of the downstream duplex must exceed a critical length for efficient formation of transcription competent open promoter complex.

Stochastic sequence-level model of coupled transcription and translation in prokaryotes

BackgroundIn prokaryotes, transcription and translation are dynamically coupled, as the latter starts before the former is complete. Also, from one transcript, several translation events occur in parallel. To study how events in transcription elongation affect translation elongation and fluctuations in protein levels, we propose a delayed stochastic model of prokaryotic transcription and translation at the nucleotide and codon level that includes the promoter open complex formation and alternative pathways to elongation, namely pausing, arrests, editing, pyrophosphorolysis, RNA polymerase traffic, and premature termination. Stepwise translation can start after the ribosome binding site is formed and accounts for variable codon translation rates, ribosome traffic, back-translocation, drop-off, and trans-translation.ResultsFirst, we show that the model accurately matches measurements of sequence-dependent translation elongation dynamics. Next, we characterize the degree of coupling between fluctuations in RNA and protein levels, and its dependence on the rates of transcription and translation initiation. Finally, modeling sequence-specific transcriptional pauses, we find that these affect protein noise levels.ConclusionsFor parameter values within realistic intervals, transcription and translation are found to be tightly coupled in Escherichia coli, as the noise in protein levels is mostly determined by the underlying noise in RNA levels. Sequence-dependent events in transcription elongation, e.g. pauses, are found to cause tangible effects in the degree of fluctuations in protein levels.

Inference of Kinetic Parameters of Delayed Stochastic Models of Gene Expression Using a Markov Chain Approximation

We propose a Markov chain approximation of the delayed stochastic simulation algorithm to infer properties of the mechanisms in prokaryote transcription from the dynamics of RNA levels. We model transcription using the delayed stochastic modelling strategy and realistic parameter values for rate of transcription initiation and RNA degradation. From the model, we generate time series of RNA levels at the single molecule level, from which we use the method to infer the duration of the promoter open complex formation. This is found to be possible even when adding external Gaussian noise to the RNA levels.