RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database of the best-known regulatory network of any free-living organism, that of Escherichia coli K-12. The major conceptual change since 3 years ago is an expanded biological context so that transcriptional regulation is now part of a unit that initiates with the signal and continues with the signal transduction to the core of regulation, modifying expression of the affected target genes responsible for the response. We call these genetic sensory response units, or Gensor Units. We have initiated their high-level curation, with graphic maps and superreactions with links to other databases. Additional connectivity uses expandable submaps. RegulonDB has summaries for every transcription factor (TF) and TF-binding sites with internal symmetry. Several DNA-binding motifs and their sizes have been redefined and relocated. In addition to data from the literature, we have incorporated our own information on transcription start sites (TSSs) and transcriptional units (TUs), obtained by using high-throughput whole-genome sequencing technologies. A new portable drawing tool for genomic features is also now available, as well as new ways to download the data, including web services, files for several relational database manager systems and text files including BioPAX format.
Transcription by RNA polymerase (RNAP) is often regulated by interactions with control proteins to link specific gene expression to environmental signals and temporal cues. Often activators help recruit RNAP to promoters to increase initiation rates (Busby and Ebright 1999). In contrast, activity of the bacterial 54 containing RNAP holoenzyme is regulated at the DNA melting step (for review, see Buck et al. 2000). Hydrolysis of an NTP by an activator drives a change in configuration of the 54 -holoenzyme, converting the initial closed complex to an open complex to allow interaction with the template DNA for mRNA synthesis (Wedel and Kustu 1995). Preopening of DNA templates does not overcome the requirement for NTP hydrolysis by an activator to promote engagement of the holoenzyme with the melted DNA (Wedel and Kustu 1995;Cannon et al. 1999).The activators of 54 -holoenzyme are members of the large AAA+ protein family, which use ATP binding and hydrolysis to remodel their substrates (Neuwald et al. 1999;Cannon et al. 2000Cannon et al. , 2001. The greater part of the central domain of 54 activators corresponds to the AAA core structure, and includes ATP-binding and hydrolyzing determinants. The 54 protein is known to be the primary target for the NTPase of activators, but how activators use NTP binding and hydrolysis is not well understood (Cannon et al. 2000). Similarly, the nature of the interaction between 54 and the activator is not well described, but an interaction with 54 can be detected in the case of the DctD activator by protein cross-linking (Lee and Hoover 1995). Here we show that the use of ADP-aluminum fluoride, an analog of ATP that mimics ATP in the transition state for hydrolysis, allows formation of a stable complex among the activator PspF, the PspF and NifA central activating domains, and 54 . The binding assay was used to help define determinants in 54 and the activator needed for their interaction, and to show that binding can lead to an altered 54 -DNA footprint. The need for a transition-state analog of ATP for protein-protein binding is discussed in relation to the required ATPase activity of activators of 54 -dependent transcription. In particular, it seems that altered functional states of activators exist as ATP is hydrolyzed. This suggests a parallel to some switch and motor proteins that use nucleotide binding and hydrolysis to establish alternate functional states (Hirose and Amos 1999).
Despite almost 40 years of molecular genetics research in Escherichia coli a major fraction of its Transcription Start Sites (TSSs) are still unknown, limiting therefore our understanding of the regulatory circuits that control gene expression in this model organism. RegulonDB (http://regulondb.ccg.unam.mx/) is aimed at integrating the genetic regulatory network of E. coli K12 as an entirely bioinformatic project up till now. In this work, we extended its aims by generating experimental data at a genome scale on TSSs, promoters and regulatory regions. We implemented a modified 5′ RACE protocol and an unbiased High Throughput Pyrosequencing Strategy (HTPS) that allowed us to map more than 1700 TSSs with high precision. From this collection, about 230 corresponded to previously reported TSSs, which helped us to benchmark both our methodologies and the accuracy of the previous mapping experiments. The other ca 1500 TSSs mapped belong to about 1000 different genes, many of them with no assigned function. We identified promoter sequences and type of σ factors that control the expression of about 80% of these genes. As expected, the housekeeping σ70 was the most common type of promoter, followed by σ38. The majority of the putative TSSs were located between 20 to 40 nucleotides from the translational start site. Putative regulatory binding sites for transcription factors were detected upstream of many TSSs. For a few transcripts, riboswitches and small RNAs were found. Several genes also had additional TSSs within the coding region. Unexpectedly, the HTPS experiments revealed extensive antisense transcription, probably for regulatory functions. The new information in RegulonDB, now with more than 2400 experimentally determined TSSs, strengthens the accuracy of promoter prediction, operon structure, and regulatory networks and provides valuable new information that will facilitate the understanding from a global perspective the complex and intricate regulatory network that operates in E. coli.
The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R 1 ) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R 1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R 1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or 16 -22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R 1 receptor, whereas domain III 16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R 1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through -16 of domain III promoting membrane insertion and cell death.
Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B . divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B . divergens . Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B . divergens genome. The new assembly shows better continuity and has a higher correspondence to B . bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B . divergens : the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B . divergens , we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis.
Babesia divergens causes significant morbidity and mortality in cattle and splenectomized or immunocompromised individuals. Here, we present a 10.7-Mb high-quality draft genome of this parasite close to chromosome resolution that will enable comparative genome analyses and synteny studies among related parasites.
Edited by Chris WhitfieldBacillus thuringiensis Cry toxins are used worldwide for controlling insects. Cry1Ab is produced as a 130-kDa protoxin that is activated by proteolytic removal of an inert 500 amino-acidlong C-terminal region, enabling the activated toxin to bind to insect midgut receptor proteins, leading to its membrane insertion and pore formation. It has been proposed that the C-terminal region is only involved in toxin crystallization, but its role in receptor binding is undefined. Here we show that the C-terminal region of Cry1Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) insect receptors. ELISA, ligand blot, surface plasmon resonance, and pulldown assays revealed that the Cry1Ab C-terminal region binds to both ALP and APN but not to cadherin. Thus, the C-terminal region provided a higher binding affinity of the protoxin to the gut membrane that correlated with higher toxicity of protoxin than activated toxin. Moreover, Cry1Ab domain II loop 2 or 3 mutations reduced binding of the protoxin to cadherin but not to ALP or APN, supporting the idea that protoxins have additional binding sites. These results imply that two different regions mediate the binding of Cry1Ab protoxin to membrane receptors, one located in domain II-III of the toxin and another in its C-terminal region, suggesting an active role of the C-terminal protoxin fragment in the mode of action of Cry toxins. These results suggest that future manipulations of the C-terminal protoxin region could alter the specificity and increase the toxicity of B. thuringiensis proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.