Positive Control Mutants Research Articles

Structural insights into the role of deleterious mutations at the dimeric interface of Toll-like receptor interferon-β related adaptor protein.

Toll-like receptors (TLRs) are major players in the innate immune system-recognizing pathogens and differentiating self/non-self components of immunity. These proteins are present either on the plasma membrane or endosome and recognize pathogens at their extracellular domains. They are characterized by a single transmembrane helix and an intracellular toll-interleukin-1 receptor (TIR) domain. Few TIRs directly invoke downstream signaling, while others require other TIR domains of adaptors like TIR domain-containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM). On recognizing pathogenic lipopolysaccharides, TLR4 dimerises and interacts with the intracellular TRAM dimer through the TIR domain to recruit a downstream signaling adaptor (TRIF). We have performed an in-depth study of the structural effect of two mutations (P116H and C117H) at the dimeric interface of the adaptor TRAM, which are known to abrogate downstream signaling. We modeled the structure and performed molecular dynamics studies in order to decipher the structural basis of this effect. We observed that these mutations led to an increased radius of gyration of the complex and resulted in several changes to the interaction energy values when compared against the wild type (WT) and positive control mutants. We identified highly interacting residues as hubs in the WT dimer, and a few such hubs that were lost in the mutant dimers. Changes in the protein residue path, hampering the information flow between the crucial A86/E87/D88/D89 and T155/S156 sites, were observed for the mutants. Overall, we show that such residue changes can have subtle but long-distance effects, impacting the signaling path allosterically.

Detection of Colistin Resistance in Pseudomonas aeruginosa Using the MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer.

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Abstract 2420: Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib

Abstract Hundreds of somatic ERBB4 mutations have been described in cancer tissues with very limited information about their functional significance. However, analyses of individual mutants have indicated that activating ERBB4 mutations, such as ERBB4 K935I, do exist. Understanding the functional consequences of ERBB4 mutations is needed in order to assess the relevance of targeting ERBB4 in human cancers with matched therapies such as neratinib. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that binds to and potently inhibits ERBB4 kinases activity in-vitro, and is currently approved clinically for the treatment of early stage and metastatic HER2+ breast cancers. We set up to address the transforming potential of individual ERBB4 variants by selecting 18 mutations (Table 1) from cBioPortal data (www.cbioportal.org) using the following criteria: 1) the mutations were recurrent, 2) they were analogous to activating mutations described for other oncogenic ERBB family members and/or 3) their position at receptor dimerization interfaces suggested functional relevance. Retroviral pBABE-puro-gateway vector was used to achieve functional, but still near-physiological expression levels of ERBB4 in different cell backgrounds. A screen for optimal cellular background to assess the transforming potential of ERBB4 variants indicated that 1) IL-3 independent growth of mouse lymphoid Ba/F3 cells, 2) focus formation analysis of mouse NIH-3T3 fibroblasts, and 3) ligand-induced proliferation of human mammary epithelial MCF-10A provided read-outs with robust differences when the effects of the positive control mutant ERBB4 K935I was compared to wild-type ERBB4. These models are now being used to address the transforming potential of the 18 mutations (Table 1). Table 1.Somatic ERBB4 mutations chosen for functional analyses.R106CE452KR711CL798RG870RK1223TS303FR524CG741EV840IG907ES1289AR393WR544WS774GR847HR992CR1304W Citation Format: Peppi Suominen, Deepankar Chakroborty, Kari J. Kurppa, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, Klaus Elenius. Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2420.

Effects of WNT1 c.110\u2009T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/\u03b2-catenin signaling pathway

BackgroundWNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI.MethodsEmpty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/β-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells.ResultsCompared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated β-catenin (non-p-β-catenin) and phosphorylated GSK-3β (p-GSK-3β) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-β-catenin or p-GSK-3β were observed in the mutation groups.ConclusionsWNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/β-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.

Gene Editing of Arabidopsis thaliana RNA Dependent RNA Polymerases Using CRISPR/Cas9

Viruses are a prolific force able to infect any form of life on the planet, including plants. Around the world, plant viruses cause considerable yield loss in many agriculture crops every year. Antiviral immunity in plants is mediated by RNA silencing and RNA decay mechanisms, which proceed as follows: once a virus inserts its genetic material into a cell, dicer‐like ribonucleases (DCLs) detect the viral RNA and fragment it into small interfering RNAs (siRNAs). These RNA fragments are then bound by Argonaute proteins and form an RNA‐induced silencing complex (RISC). The RISC binds to complementary sequences of the viral RNA and remains bound, thus deactivating or degrading the fragment by enzymatically cleaving the RNA. siRNA antiviral RNA silencing is utilized for acute or cellular antiviral defense, but it also plays a role in systemic antiviral defense. In plants the systemic silencing mechanism is mediated by enzymes called RNA‐dependent RNA polymerases (RDR) that synthesize double stranded RNA (dsRNA) from single strand RNA. These dsRNAs are then processed through the same siRNA‐silencing pathway, producing new siRNAs that can silence viral genes. The supplementary siRNAs can then be transported between cells through the plants’ vasculature and the plasmodesmata. This leads to systemic treatment and defense in the plant. Though the core genetic components of antiviral RNA silencing have been determined, additional components remain to be identified and characterized. For example, the A. thaliana genome encodes six RDR genes. It is known that RDR1 and RDR6 have major roles in antiviral RNA silencing and that RDR2 mediates biogenesis of cellular siRNAs. However, the role of the RDR3, RDR4, and RDR5 genes is not known. Though single mutants of RDR3, RDR4, and RDR5, have been constructed, double or triple mutants cannot be obtained by tDNA mutation because all three genes are linked together on the second chromosome. Our hypothesis was that RDR3 and RDR4 can be inactivated by site‐specific genome editing using the CRISPR/Cas9 technique to develop mutants lacking these genes. Further experiments show the expression of the Cas9 protein in the second generation, confirming transformation of A. thaliana proteins and RNA. We expect that creating this sextuple mutant, in which all six RDR genes are deactivated, allows the continued exploration of the role of RDRs in antiviral silencing. The RDR3 and RDR4 inactive mutants will also facilitate the necessary positive and negative control mutants needed for future analytical studies.Support or Funding InformationUndergraduate Creative Activities and Research Experience Project, funded by the PepsiQuasi Endowment and Union Bank & Trust. National Institute of Health (R01GM120108). Institute of Agriculture and Natural Resources, Agricultural Research Division, Undergraduate Student Research ProgramCRISPR single guide RNAs.A) Target Sites and sgRNAs in RDR3a an dRDR3b found using CCTOP (Stemmer et al. 2015). Small guide RNAs are the target sites minus the PAM colored in red. B) Single guide RNAs targeting RDR3a and RDR3b.Figure 1

Importance of kelch 13 C580Y mutation in the studies of artemisinin resistance in Plasmodium falciparum in Greater Mekong Subregion.

The mortality caused by Plasmodium falciparum was reduced by Artemisinin (ART) and ART combination therapy (ACT). However, Artemisinin resistance (ART-R) emerge during 2008 in Cambodia and spread to Greater Mekong Subregion (GMS). ART-R was confirmed not to spread to India, a gateway to whole Africa. The whole genome sequencing approach of P. falciparum assumed the k13 gene encoded Kelch protein was discovered to be associated with ART-R. Of the single nucleotide polymorphisms (SNPs) of k13 gene, C580Y mutant was commonly dominant in Cambodia, Myanmar, Thailand, Laos and Vietnam and assumed to be one of strong molecular markers for ART-R in P. falciparum isolates in GMS. Literatures published between 2017 and 2018 were reviewed in this work. F446I is observed to be doubtful molecular marker as ART-R marker. Transgenic experiment showed that parasite with F446I mutation displayed prolonged clearance in respond to ART while C580Y was applied as positive control mutant. Furthermore, study of C580Y allele in four countries Cambodia, Thailand, Laos resulted in single origin whereas the parasite with this allele showed multi-origin in three Provinces of Vietnam. As artemisinin was short acting drug, the role of long acting partner drug was studied by using transgenic C580Y mutant and C580 to leave recrudescent P. falciparum. Recently, there was treatment failure with ACT in some countries in GMS. In this review, the importance of C580Y mutation in the study of ART-R was discussed.

Mutations in β′ subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the β' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the β' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the β' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.

Protein–Protein Interactions Between σ 70 Region 4 of RNA Polymerase and Escherichia coli SoxS, a Transcription Activator That Functions by the Prerecruitment Mechanism: Evidence for “Off-DNA” and “On-DNA” Interactions

According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the α subunit's C-terminal domain plays a role in activating both classes of promoter by making protein–protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of σ 70 (σ 70 R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these σ 70 R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and σ 70 R4 can interact in solution (i.e., “off-DNA”). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of σ 70 R4 that reduce activation in E. coli also reduce the SoxS–σ 70 R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur “on-DNA” at class II promoters. Finding that SoxS contacts σ 70 R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein–protein contacts with two different subunits of RNAP.

N‐ and C‐terminal regions of the quorum‐sensing activator TraR cooperate in interactions with the alpha and sigma‐70 components of RNA polymerase

Positive control (PC) mutants defining 20 residues of the quorum-sensing activator TraR were isolated that bind DNA but show defects in activating transcription from class I, class II or both types of promoters. These PC residues, located in both the N- and C-terminal regions, combine to form three patches, one on the top (II) and two near the DNA binding domain on both lateral faces of the dimer (I and III). Patches I and II, but not patch III, involve residues from both protomers and are essential for activation. TraR-mediated activation in Escherichia coli requires expression of the alpha-subunit of Agrobacterium (alpha(At)). We report that TraR also activates a class II promoter in E. coli when coexpressed with sigma(70)(At). Analyses in E. coli expressing alpha(At), sigma(70)(At) or both subunits indicate that most of the PC residues are important for interactions with alpha(At) and that these interactions are predominant for activation of class II promoters. Using the E. coli system we identified nine residues in the C-terminal domain of alpha(At) that are required for stimulating TraR-mediated activation. We conclude that N- and C-terminal residues of TraR from both protomers cooperate to define regions of the protein important for interactions with RNAP.

Dual Role for Transactivator Protein C in Activation of mom Promoter of Bacteriophage Mu

Transactivator C protein of bacteriophage Mu activates the mom gene of the phage by an unusual mechanism. DNA binding by C to its site results in unwinding of the neighboring sequences, realigning the out-of-phase promoter elements to facilitate RNA polymerase (RNAP) binding. High level stimulation of a C-independent constitutive promoter mutant (where RNAP is already bound) by the transactivator suggested an additional mechanism of transcription activation at a step after RNAP recruitment. In this study, we have investigated the various steps of promoter-polymerase interactions during transcription initiation by using both the promoter mutant and a positive control (pc) mutant of C protein. The transactivator does not influence formation of the open complex or its stability after facilitating the RNAP binding. However, at a subsequent step, the protein exerts an important role, enhancing the promoter clearance by increasing the productive RNAP.promoter complex. The pc mutant of the transactivator C is compromised at this step, supporting the additional downstream role for C in mom transcription activation. We suggest that this unusual multistep activation of Pmom has evolved to ensure irreversibility of the switch during the late lytic cycle of the phage.

Identification of the CRP regulon using in vitro and in vivo transcriptional profiling

The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gene promoters. In this study, we have exploited a newly developed technique, run-off transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using ROMA, we identified 176 operons that were activated by CRP in vitro and 16 operons that were repressed. Using positive control mutants in different regions of CRP, we were able to classify the different promoters into class I or class II/III. A total of 104 operons were predicted to contain Class II CRP-binding sites. Sequence analysis of the operons that were repressed by CRP revealed different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional profiles failed to identify most CRP-dependent regulation because of the complexity of the regulatory network. Analysis of these operons supports the hypothesis that CRP is not only a regulator of genes required for catabolism of sugars other than glucose, but also regulates the expression of a large number of other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons.

Genomic studies with Escherichia coli MelR protein: applications of chromatin immunoprecipitation and microarrays.

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

Domains Formed within the N-terminal Region of the Quorumsensing Activator TraR Are Required for Transcriptional Activation and Direct Interaction with RpoA from Agrobacterium

TraR, a quorum-sensing activator, induces transcription from its binding site, the tra-box, located upstream of Ti plasmid target promoters. TraR activated expression of a lacZ reporter in Escherichia coli only when RpoAAt from Agrobacterium tumefaciens was co-expressed. As assessed by gel retardation assays RpoAAt, but not RpoAEc, formed a ternary complex with TraR and a tra-box probe in vitro. TraR formed similar ternary complexes with alphaCTDAt but not with NTDAt, the C- and N-terminal segments of RpoAAt. As measured by surface plasmon resonance refractometry, TraR interacted directly with RpoAAt with an affinity about five times greater than that observed for its interaction with RpoAEc. The activator interacted with alphaCTDAt with kinetics and affinities similar to those of the full-sized -subunit. Positive control (PC) mutations at Asp-10 and Gly-123 of TraR did not affect DNA binding but greatly decreased the TraR-RpoAAt interaction. These two residues combine to form two patches on the activator, one of which may be involved in interaction with RpoA. When co-expressed, mutants of TraR with substitutions at Asp-10 complementing mutants with substitutions at Gly-123 for gene activation in an allele-specific manner. Co-expression studies with TraR and its PC mutants, and also with complementary PC alleles of TraR, coupled with three-dimensional structure are consistent with a hypothesis that both Asp-10/Gly-123 patches are required for activator function.

Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Tests Workgroup Report—Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.

A Comprehensive Alanine Scanning Mutagenesis of the Escherichia coli Transcriptional Activator SoxS: Identifying Amino Acids Important for DNA Binding and Transcription Activation

SoxS is the direct transcriptional activator of the superoxide regulon. SoxS recognizes a highly degenerate “soxbox” DNA sequence, and activates transcription from class I and class II promoters. SoxS is the smallest member of the AraC/XylS family of transcription regulators whose hallmark is dual helix-turn-helix (HTH) DNA-binding motifs. Evidence suggests that the N-terminal HTH motif of SoxS interacts with a highly conserved region of the soxbox termed recognition element 1 (RE1), while the C-terminal HTH motif interacts with the less conserved recognition element 2 (RE2). In the work described here, we prepared a complete library of 101 SoxS mutants containing single alanine substitutions of SoxS, and we characterized the mutant proteins in vivo and in vitro. With SoxS being closely related to MarA, we analyzed the effects of the SoxS mutations in the context of the MarA– mar crystal structure and with respect to the NMR study of MarA–DNA complexes in solution. From the properties of the alanine substitutions, we conclude the following. (1) Surface-exposed residues of helix 3 and helix 6, the recognition helices of the dual HTH motifs, are important to DNA binding and transcription activation; however, substitutions of residues predicted from the MarA– mar crystal structure to make contact with the sugar–phosphate backbone are more detrimental to DNA binding than mutations predicted to make base-specific contacts. (2) Substitution of several residues within the recognition helix predicted to make base-specific contacts with RE2 have relatively little effect on DNA-binding, suggesting the possibility of alternative protein–DNA interactions than those inferred from the MarA– mar crystal structure. (3) DNA binding and transcription activation were reduced by substitution of conserved amino acid residues comprising the hydrophobic core, presumably because they disrupt the structural integrity of SoxS. (4) Mutant K30A appears to be a positive control mutant defective in a protein–protein interaction with RNA polymerase that is required for transcription activation at all SoxS-dependent promoters because it binds and bends DNA normally but fails to activate transcription from both classes of promoters. Alanine substitutions of surface-exposed residues H3, K5, D9, S31, and V45 confer a similar phenotype. Since these residues are near K30 on the surface of the protein, the surface formed by the six residues may be used to make protein–protein interactions with RNA polymerase that are required for transcription activation at both class I and class II SoxS-dependent promoters. (5) Mutants F74A, D75A, M78A, D79A and Q85A appear to define a surface required for protein–protein interaction with RNA polymerase specifically at class II promoters because these positive control mutants bind and bend DNA normally but are defective in activation of class II promoters but not class I promoters. These SoxS mutants that bind and bend DNA normally but are defective in transcription activation represent the first positive control mutants with putative defects in protein–protein interactions with RNA polymerase among the SoxS/MarA/Rob subset of the AraC/XylS family of transcription regulators.

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Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations.

Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the sigma(70) subunit. A motA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five sigma(70) mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant sigma(70) proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The sigma(70) substitutions affected the 4.2 region, which binds the -35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. coli promoters. This defect could not be explained solely by a disruption in -35 recognition since similar results were obtained with extended -10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

Quorum sensing in Vibrio fischeri: analysis of the LuxR DNA binding region by alanine-scanning mutagenesis.

LuxR is the transcriptional activator for quorum-sensing control of luminescence in Vibrio fischeri. A series of alanine-scanning mutants spanning a predicted helix-turn-helix region in the DNA binding domain of LuxR was constructed, and the activity of each of the LuxR mutant proteins in recombinant Escherichia coli was investigated. The region covered by the mutagenesis spanned residues 190 to 224. About half of the alanine-scanning mutants showed activities similar to that of the wild-type LuxR: at least two were positive-control mutants, four appeared to be defective in DNA binding, and several others were characterized as DNA binding affinity mutants. This analysis, taken together with information about other bacterial transcription factors, provides insights into amino acid residues in LuxR that are involved in DNA binding and transcriptional activation.

Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3

At Class II CRP-dependent promoters, the Escherichia coli cyclic AMP receptor protein (CRP) activates transcription by making multiple interactions with RNA polymerase (RNAP). Two discrete surfaces of CRP, known as Activating Region 1 (AR1) and Activating Region 2 (AR2), interact with the C-terminal and N-terminal domains, respectively, of the α subunit of RNAP. Activating Region 3 (AR3) is a third separate surface of CRP, which is thought to interact with a target in the C-terminal region of the RNAP σ70 subunit. We have used a CRP mutant that functions primarily via AR3, CRP HL159 KE101 KN52, as a tool to identify residues within AR3 that are important for activation. This was achieved by screening a random mutant library of the gene encoding CRP HL159 KE101 KN52 for positive control mutants at Class II CRP-dependent promoters, and also by performing alanine scanning mutagenesis. Using both in vivo reporter assays and in vitro transcription assays, we measured the effects of key substitutions within AR3 on transcription activation in both CRP HL159 KE101 KN52 and wild-type CRP. We show that a cluster of negatively charged surface-exposed residues at positions 53, 54, 55 and 58 is required for optimal activation at a Class II, but not at a Class I, CRP-dependent promoter. We conclude that these residues in AR3 of CRP form an activatory determinant for Class II transcription activation. Abortive initiation assays were used to show that this activatory determinant accelerates the rate of isomerisation from the closed to open complex at a Class II CRP-dependent promoter. AR3 of CRP also contains an inhibitory determinant: the lysine residue at position 52 of CRP is inhibitory to maximal levels of transcription activation from Class II promoters. We show that the negative effects of K52 are not simply due to “masking” of the negatively charged residues at positions 53, 54, 55 and 58. Our results suggest that, during activation by wild-type CRP, the activatory and inhibitory determinants of AR3 balance each other. Thus, activation is predominantly determined by AR1 and AR2.

Suppression analysis of positive control mutants of NifA reveals two overlapping promoters for Klebsiella pneumoniae rpoN

Activation of gene expression relies on direct molecular interactions between the RNA polymerase and transcription factors. Eubacterial enhancer-binding proteins (EBPs) activate transcription by binding to distant sites and, simultaneously, contacting the σ 54-holoenzyme form of the RNA polymerase (Eσ 54). The interaction between the EBP and Eσ 54is transient, such that it has been difficult to be studied biochemically. Therefore, the details of this molecular recognition event are not known. Genetic and physical evidences suggest that the highly conserved C3 region in the activation domain of the EBP has major determinants for positive control and for the interaction with Eσ 54. To further investigate the target of this region we searched for extragenic suppressors of some C3 region mutant derivatives of NifA. As a first step we mutagenized Klebsiella pneumoniae rpoN, the gene that codes for σ 54. A mutant allele, rpoN 1320, that suppressed two different NifA derivatives was obtained. Immunodetection of σ 54 and transcriptional initiation studies demonstrated that the cause of the suppression was an enhanced expression of rpoN. A single point mutation was responsible for the phenotype. It mapped at the −10 region of an unidentified promoter, here denominated rpoN p1, and increased its similarity to the consensus. A second upstream promoter, denominated rpoN p2, was also identified. Its −10 region partially overlaps with the −35 region of rpoN p1. Interestingly, the promoter-up −10 mutation in rpoN p1 caused a reduction in the expression from rpoN p2, likely reflecting a stronger occupancy of the former promoter by the RNA polymerase at the expense of the latter. The presence of two overlapping promoters competing for the RNA polymerase implies a complex regulatory pattern that needs elucidation. The fact that increasing the concentration of σ 54 in the cell can suppress positive control mutants of NifA adds further evidence for their direct interaction in the activation process.