scholarly journals Principles of gene regulation quantitatively connect DNA to RNA and proteins in bacteria

2021 ◽  
Author(s):  
Rohan Balakrishnan ◽  
Matteo Mori ◽  
Igor Segota ◽  
Zhongge Zhang ◽  
Ruedi Aebersold ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Growth Conditions ◽  
Global Constraints ◽  
Genetic Circuits ◽  
Cellular Functions ◽  
E Coli ◽  
Gene Regulatory ◽  
Protein Density ◽  
Transcriptional Output ◽  
Ribosomal Activity

Bacteria allocate their proteome to cellular functions differently in different growth conditions. It is largely unknown how such allocation arises from known mechanisms of gene regulation while constrained by limited translation capacity and fixed protein density. Here, we performed absolute transcriptomic and proteomic analysis for E. coli across many conditions, obtaining a plethora of results on promoters and mRNAs characteristics that clash with conventional expectations: the majority of mRNAs exhibit similar translational efficiencies, while the promoter strengths are vastly different across genes. These characteristics prescribe two principles of gene regulation guiding bacteria to attain the desired protein allocation under global constraints: Total transcriptional output is tightly coordinated with ribosomal activity, and the concentrations of individual proteins are largely set by transcription. These two principles lead to a quantitative formulation of Central Dogma which unravels the complex relationship between gene regulatory activities and mRNA/protein concentrations across conditions. The knowledge obtained will be invaluable for accurately inferring gene regulatory interactions from 'omics data, as well as for guiding the design of genetic circuits for synthetic biology applications in E. coli and other organisms.

scholarly journals Model-guided design of mammalian genetic programs

2021 ◽  
Vol 7 (8) ◽  
pp. eabe9375
Author(s):  
J. J. Muldoon ◽  
V. Kandula ◽  
M. Hong ◽  
P. S. Donahue ◽  
J. D. Boucher ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Computational Models ◽  
Biological Complexity ◽  
Genetic Circuits ◽  
Cellular Functions ◽  
High Performing ◽  
Design Objective ◽  
Complex Functions ◽  
Mammalian Genetic ◽  
Analog Processing

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals Bacterial growth laws reflect the evolutionary importance of energy efficiency

2014 ◽  
Vol 112 (2) ◽  
pp. 406-411 ◽  
Author(s):  
Arijit Maitra ◽  
Ken A. Dill
Keyword(s):  
Energy Efficiency ◽  
Bacterial Growth ◽  
Growth Conditions ◽  
High Energy ◽  
Fast Growth ◽  
Growth Speed ◽  
Energy Costs ◽  
Extensive Literature ◽  
E Coli ◽  
Growth Laws

We are interested in the balance of energy and protein synthesis in bacterial growth. How has evolution optimized this balance? We describe an analytical model that leverages extensive literature data on growth laws to infer the underlying fitness landscape and to draw inferences about what evolution has optimized inEscherichia coli. IsE. colioptimized for growth speed, energy efficiency, or some other property? Experimental data show that at its replication speed limit,E. coliproduces about four mass equivalents of nonribosomal proteins for every mass equivalent of ribosomes. This ratio can be explained if the cell’s fitness function is the the energy efficiency of cells under fast growth conditions, indicating a tradeoff between the high energy costs of ribosomes under fast growth and the high energy costs of turning over nonribosomal proteins under slow growth. This model gives insight into some of the complex nonlinear relationships between energy utilization and ribosomal and nonribosomal production as a function of cell growth conditions.

Galactokinase Gene Fusion in the Study of Gene Regulation in E. Coli, Streptomyces, Yeast, and Higher Cell Systems

1986 ◽  
pp. 151-180 ◽  
Author(s):  
Martin Rosenberg ◽  
Mary Brawner ◽  
Jessica Gorman ◽  
Mitchell Reff
Keyword(s):  
Gene Regulation ◽  
Gene Fusion ◽  
Cell Systems ◽  
E Coli

scholarly journals A MATLAB Toolbox for Modeling Genetic Circuits in Cell-Free Systems

2020 ◽  
Author(s):  
Vipul Singhal ◽  
Zoltan A. Tuza ◽  
Zachary Z. Sun ◽  
Richard M. Murray
Keyword(s):  
Reaction Rate ◽  
Behavior Prediction ◽  
Genetic Circuits ◽  
Matlab Toolbox ◽  
Parameter Inference ◽  
Batch Mode ◽  
E Coli ◽  
Entire Duration ◽  
Resource Loading

AbstractWe introduce a MATLAB based simulation toolbox, called txtlsim, for an E. coli based Transcription-Translation (TX-TL) system. This toolbox accounts for several cell-free related phenomena, such as resource loading, consumption, and degradation, and in doing so, models the dynamics of TX-TL reactions for the entire duration of batch-mode experiments. We use a Bayesian parameter inference approach to characterize the reaction rate parameters associated with the core transcription, translation and mRNA degradation mechanics of the toolbox, allowing it to reproduce constitutive mRNA and protien expression trajectories. We demonstrate the use of this characterized toolbox in a circuit behavior prediction case study for an incoherent feed-forward loop.

scholarly journals Decoding the Epitranscriptional Landscape from Native RNA Sequences

2018 ◽  
Author(s):  
Thidathip Wongsurawat ◽  
Piroon Jenjaroenpun ◽  
Trudy M. Wassenaar ◽  
Taylor D Wadley ◽  
Visanu Wanchai ◽  
...  
Keyword(s):  
Growth Conditions ◽  
Untranslated Regions ◽  
Metabolic Adaptation ◽  
Rna Modifications ◽  
Rna Sequences ◽  
Rna Methylation ◽  
E Coli ◽  
Oxford Nanopore ◽  
G Quadruplex ◽  
Bioinformatic Tool

AbstractSequencing of native RNA and corresponding cDNA was performed using Oxford Nanopore Technology. The % Error of Specific Bases (%ESB) was higher for native RNA than for cDNA, which enabled detection of ribonucleotide modification sites. Based on %ESB differences of the two templates, a bioinformatic tool ELIGOS was developed and applied to rRNAs of E. coli, yeast and human cells. ELIGOS captured 91%, 95%, ∼75%, respectively, of the known variety of RNA methylation sites in these rRNAs. Yeast transcriptomes from different growth conditions were also compared, which identified an association between metabolic adaptation and inferred RNA modifications. ELIGOS was further applied to human transcriptome datasets, which identified the well-known DRACH motif containing N6-methyadenine being located close to 3’-untranslated regions of mRNA. Moreover, the RNA G-quadruplex motif was uncovered by ELIGOS. In summary, we have developed an experimental method coupled with bioinformatic software to uncover native RNA modifications and secondary-structures within transcripts.

scholarly journals Comparative characterisation of Plasmodium falciparum Hsp70-1 relative to E. coli DnaK reveals functional specificity of the parasite chaperone

2020 ◽  
Author(s):  
Charity Mekgwa Lebepe ◽  
Pearl Rutendo Matambanadzo ◽  
Xolani Henry Makhoba ◽  
Ikechukwu Achilonu ◽  
Tawanda Zininga ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Chaperone ◽  
Chimeric Protein ◽  
Cellular Functions ◽  
E Coli ◽  
Adenosylmethionine Decarboxylase ◽  
Comparative Structure ◽  
Functional Features ◽  
Self Association

ABSTRACTHsp70 is one of the most prominent molecular chaperones. Although Hsp70s from various organisms are generally conserved, they exhibit specialised cellular functions. It remains to be fully understood how these highly conserved molecules exhibit specialised functional features. Plasmodium falciparum Hsp70-1 (PfHsp70-1) is a cytosol localised molecular chaperone that is implicated in the cyto-protection and pathogenicity of the malaria parasite. In the current study, we investigated the comparative structure-function features of PfHsp70-1 relative to its homologue, E. coli Hsp70 (DnaK) and a chimeric protein, KPf, that was constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of all the three Hsp70s exhibited similar secondary and tertiary structural fold. We further established that compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. A recombinant P. falciparum Hsp40 (PfHsp40) stimulated the ATPase activities of all the three Hsp70s. In addition, both PfHsp70-1 and KPf exhibited preference for asparagine rich peptides as opposed to DnaK. Furthermore, all the three proteins exhibited self-association capabilities in vitro. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. On the other hand, co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

Sign in / Sign up

Export Citation Format

Share Document