scholarly journals A TetR-family protein activates transcription from a new promoter motif associated with essential genes for autotrophic growth in acetogens

2019 ◽  
Author(s):  
Renato de Souza Pinto Lemgruber ◽  
Kaspar Valgepea ◽  
Ricardo Axayacatl Gonzalez Garcia ◽  
Christopher de Bakker ◽  
Robin William Palfreyman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Essential Genes ◽  
Autotrophic Growth ◽  
Reporter Protein ◽  
Protein Assay ◽  
Protein Protein Interaction ◽  
Promoter Motif ◽  
Cell Factories ◽  
Fermenting Bacteria

AbstractAcetogens can fix carbon (CO or CO2) into acetyl-CoA via the Wood-Ljungdahl pathway (WLP) that also makes them attractive cell factories for the production of fuels and chemicals from waste feedstocks. Although most biochemical details of the WLP are well understood and systems-level characterisation of acetogen metabolism has recently improved, key transcriptional features such as promoter motifs and transcriptional regulators are still unknown in acetogens. Here, we use differential RNA-sequencing to identify a previously undescribed promoter motif associated with essential genes for autotrophic growth of the model-acetogen Clostridium autoethanogenum. RNA polymerase was shown to bind to the new promoter motif using a DNA-binding protein assay and proteomics enabled the discovery of four candidates to potentially function directly in control of transcription of the WLP and other key genes of C1 fixation metabolism. Next, in vivo experiments showed that a TetR-family transcriptional regulator (CAETHG_0459) and the housekeeping sigma factor (σA) activate expression of a reporter protein (GFP) in-frame with the new promoter motif from a fusion vector in E. coli. Lastly, a protein-protein interaction assay with the RNA polymerase (RNAP) shows that CAETHG_0459 directly binds to the RNAP. Together, the data presented here advance the fundamental understanding of transcriptional regulation of C1 fixation in acetogens and provide a strategy for improving the performance of gas-fermenting bacteria by genetic engineering.

scholarly journals Rhodobacter capsulatus nifA1 Promoter: High-GC −10 Regions in High-GC Bacteria and the Basis for Their Transcription

2004 ◽  
Vol 186 (3) ◽  
pp. 740-749 ◽  
Author(s):  
Cynthia L. Richard ◽  
Animesh Tandon ◽  
Robert G. Kranz
Keyword(s):  
Rna Polymerase ◽  
Rhodobacter Capsulatus ◽  
Transcription Start Sites ◽  
E Coli ◽  
Protein Protein Interaction ◽  
Enhancer Binding Protein ◽  
Pribnow Box ◽  
Rna Polymerase Subunits

ABSTRACT It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R. capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter. We have tested the hypothesis that this activity is due to the high G+C content of the −10 sequence. A comparative analysis of R. capsulatus and other α-proteobacterial promoters with known transcription start sites suggests that the G+C content of the −10 region is higher than that for E. coli. Both in vivo and in vitro results obtained with nifA1 promoters with −10 and/or −35 variations are reported here. A major conclusion of this study is that α-proteobacteria have evolved a promiscuous sigma factor and core RNA polymerase that can transcribe promoters with high-GC −10 regions in addition to the classic E. coli Pribnow box. To facilitate studies of R. capsulatus transcription, we cloned and overexpressed all of the RNA polymerase subunits in E. coli, and these were reconstituted in vitro to form an active, recombinant R. capsulatus RNA polymerase with properties mimicking those of the natural polymerase. Thus, no additional factors from R. capsulatus are necessary for the recognition of high-GC promoters or for activation by R. capsulatus NtrC. The addition of R. capsulatus σ70 to the E. coli core RNA polymerase or the use of −10 promoter mutants did not facilitate R. capsulatus NtrC activation of the nifA1 promoter by the E. coli RNA polymerase. Thus, an additional barrier to activation by R. capsulatus NtrC exists, probably a lack of the proper R. capsulatus NtrC-E. coli RNA polymerase (protein-protein) interaction(s).

scholarly journals A TetR-Family Protein (CAETHG_0459) Activates Transcription From a New Promoter Motif Associated With Essential Genes for Autotrophic Growth in Acetogens

2019 ◽  
Vol 10 ◽  
Author(s):  
Renato de Souza Pinto Lemgruber ◽  
Kaspar Valgepea ◽  
Ricardo Axayacatl Gonzalez Garcia ◽  
Christopher de Bakker ◽  
Robin William Palfreyman ◽  
...  
Keyword(s):  
Essential Genes ◽  
Autotrophic Growth ◽  
Promoter Motif ◽  
Family Protein

In Silico Identification of a Potent Arsenic Based Approved Drug Darinaparsin against SARS-CoV-2: Inhibitor of RNA Dependent RNA polymerase (RdRp) and Essential Proteases

2020 ◽  
Vol 20 ◽  
Author(s):  
Trinath Chowdhury ◽  
Gourisankar Roymahapatra ◽  
Santi M. Mandal
Keyword(s):  
Rna Polymerase ◽  
Viral Entry ◽  
In Silico ◽  
Rna Dependent Rna Polymerase ◽  
Replication Complex ◽  
In Silico Study ◽  
Life Threatening ◽  
In Vivo Analysis ◽  
3Cl Protease

Background: COVID-19 is a life threatening novel corona viral infection to our civilization and spreading rapidly. Terrific efforts are generous by the researchers to search for a drug to control SARS-CoV-2. Methods: Here, a series of arsenical derivatives were optimized and analyzed with in silico study to search the inhibitor of RNA dependent RNA polymerase (RdRp), the major replication factor of SARS-CoV-2. All the optimized derivatives were blindly docked with RdRp of SARS-CoV-2 using iGEMDOCK v2.1. Results: Based on the lower idock score in the catalytic pocket of RdRp, darinaparsin (-82.52 kcal/mol) revealed most effective among them. Darinaparsin strongly binds with both Nsp9 replicase protein (-8.77 kcal/mol) and Nsp15 endoribonuclease (-8.3 kcal/mol) of SARS-CoV-2 as confirmed from the AutoDock analysis. During infection, the ssRNA of SARS-CoV2 is translated into large polyproteins forming viral replication complex by specific proteases like 3CL protease and papain protease. This is also another target to control the virus infection where darinaparsin also perform the inhibitory role to proteases of 3CL protease (-7.69 kcal/mol) and papain protease (-8.43 kcal/mol). Conclusion: In host cell, the furin protease serves as a gateway to the viral entry and darinaparsin docked with furin protease which revealed a strong binding affinity. Thus, screening of potential arsenic drugs would help in providing the fast invitro to in-vivo analysis towards development of therapeutics against SARS-CoV-2.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals In vivo interactome profiling by enzyme‐catalyzed proximity labeling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yangfan Xu ◽  
Xianqun Fan ◽  
Yang Hu
Keyword(s):  
State Of The Art ◽  
Catalytic Efficiency ◽  
Biological Processes ◽  
Protein Protein Interaction ◽  
Current State ◽  
Protein Interactome ◽  
Potential Applications ◽  
Protein Protein Interaction Networks ◽  
Temporal And Spatial

AbstractEnzyme-catalyzed proximity labeling (PL) combined with mass spectrometry (MS) has emerged as a revolutionary approach to reveal the protein-protein interaction networks, dissect complex biological processes, and characterize the subcellular proteome in a more physiological setting than before. The enzymatic tags are being upgraded to improve temporal and spatial resolution and obtain faster catalytic dynamics and higher catalytic efficiency. In vivo application of PL integrated with other state of the art techniques has recently been adapted in live animals and plants, allowing questions to be addressed that were previously inaccessible. It is timely to summarize the current state of PL-dependent interactome studies and their potential applications. We will focus on in vivo uses of newer versions of PL and highlight critical considerations for successful in vivo PL experiments that will provide novel insights into the protein interactome in the context of human diseases.

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