Download or read book Mutation and Modulation of Translational Machinery in E Coli written by Corey M. Dambacher and published by . This book was released on 2011 with total page 366 pages. Available in PDF, EPUB and Kindle. Book excerpt: The prokaryotic translational machinery is comprised of multiple components, including the ribosome, and aminoacyl tRNA synthetase enzymes. The roles of various ribosomal proteins have been studied for several decades, however many of these proteins are encoded by essential genes, making studies of ribosomal proteins very difficult. Many of the ribosomal proteins are involved in auto-regulatory networks, and direct assembly of the ribosome. Primary binding proteins nucleate assembly, while secondary and tertiary binding proteins can be required for passage from non-productive assembly intermediates to native assembly conformations. Studies of assembly have led to an increased understanding of the pathway in which this macromolecule is formed, but these data have been primarily obtained using in vitro reconstitution experiments. More recent studies have revealed a variety of intermediates in vivo that have not been seen in vitro. In vivo experiments designed to identify new intermediates are limited to over-expression analysis, as many of the essential ribosomal proteins when deleted, do not give rise to viable cells. Additionally, existing induction systems only allow for one to induce expression of target proteins, while to study the prokaryotic ribosome in the absence of individual ribosomal proteins, one would need to shut off expression in mid-growth phase. Here we report the generation of 41 E. coli ribosomal deletion strains that enable facile studies of all essential ribosomal proteins in this organism. To address dynamic repression and induction of these proteins, we have generated a bi-directional semi-oscillatory repression/induction switch (using an unnatural aminoacyl tRNA synthetase). Taken together, these results will allow investigators to access the entire ribosomal protein network for in vivo studies of regulation and ribosomal assembly.

Download or read book Microbial Regulation of Translation written by Assaf Katz and published by Frontiers Media SA. This book was released on 2021-01-07 with total page 137 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Mutations Affecting Translation in Escherichia Coli written by Kaj Bohman and published by . This book was released on 1984 with total page 29 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Effects of Endogenous E Coli Molecules on Translational Fidelity written by Piotr Lagod and published by . This book was released on 2021 with total page 68 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribosomes translate messenger RNA (mRNA) three nucleotides at a time until translation is terminated at a stop codon. However, during all translation, frameshifting can occur, leading to the formation of proteins with amino acid sequences that differ from the in-frame product. Spontaneous frameshifting can be harmful to an organism. For instance, antibiotics such as streptomycin inhibit bacterial growth by increasing misreading and frameshifting. However, programmed translational frameshifting (which can induce high levels of frameshifting) can be used in some instances to control the ratio of specific proteins (as seen with the dnaX gene) or to increase the density of genomic information. This study explored the effects of endogenous small molecules on the IS3-frameshift-motif that is found in the transposase genes of many mobile elements. Using a cell-free protein synthesis system and a luminescent frameshift reporter, it was discovered that the addition of a small molecule extract derived from E. coli significantly decreased frameshifting, suggesting that it contains molecules that can alter translational fidelity. These experiments also revealed that the addition of the translation inhibitor chloramphenicol to translation assays at sub-inhibitory concentrations, reduced frameshift efficiency. During the studies, the role of the stability of luminescent protein reporters on the reported frameshifting levels was also explored, which is omitted in many studies. Finally, a method was developed that allows for the isolation of molecules that weakly associate with ribosomes, which opens the door for more detailed investigations of chemicals that alter translational fidelity. In conclusion, these studies provide new insight on the potential modulation of translational frameshifting by endogenous small molecules, and they set the stage to reveal the important players in this important biochemical process.

Download or read book Synthetic Metabolic Pathways written by Michael Krogh Jensen and published by Humana Press. This book was released on 2017-11-24 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume outlines key steps associated with the design, building, and testing of synthetic metabolic pathways for optimal cell factory performance and robustness, and illustrates how data-driven learning from these steps can be used for rational cost-effective engineering of cell factories with improved performance. Chapters are divided into four sections focusing on the four steps of the iterative design-build-test-learn cycle related to modern cell factory engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Synthetic Metabolic Pathways: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Download or read book Microbial Cell Factories written by Deepansh Sharma and published by CRC Press. This book was released on 2018-03-22 with total page 333 pages. Available in PDF, EPUB and Kindle. Book excerpt: Microbial Cell Factories is a conceptual, reference-based source including chapters covering microbial cell factories for industrial developments, microbial biotechnology, sustainable environmental solutions, agriculture practices, microorganisms in food processing, metabolites as next generation food additives/food processing, and microbial cell factories in alternative energy fuel generation. The book highlights trends and developments in the field of microbial products, written by an international team of leading academic and research scholars. Key Selling Features: Highlights trends and developments in microbial biotechnology Systematically reviews microbial cell factories Explores the potential of microbial cell derived industrial production Synthesizes information on environmental and agricultural uses of microbial biotechnology Contributions from an international team of leading scholars

Download or read book Effects of Ultraviolet Radiation on the Translational Machinery of E Coli written by Charles T. Backus and published by . This book was released on 1980 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Functional Mapping of the Components of the E Coli Translational Machinery written by Sonisilpa Mohapatra and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial cells generally lack membrane-bound organelles. Therefore, traditionally they were viewed as bags of freely diffusing macromolecules devoid of any internal organization. The development in imaging techniques in the last two decades has enabled better visualization of bacterial cells and led to careful investigation of their internal organization. Consequently, they are now shown to have an intricate internal organization of various proteins in their cytoplasmic space despite the absence of membrane boundaries. The spatiotemporal control of various cellular processes in bacteria is possibly regulated by this internal organization. Most cellular processes are regulated by complex networks of intermolecular interactions. A systematic study quantifying the correlation in spatial organization of various biomolecules might help to unravel their underlying interactions. A positive correlation might suggest binding to a common site or common sites of production, degradation or action. A negative correlation might suggest a mechanism that sequesters the molecules from each other. A commonly used method to quantify linear correlation between images of two different species is the Pearson Correlation Coefficient (PCC). PCC however fails both qualitatively and quantitatively in spherocylindrical cells such as E. coli. A modified PCC (MPCC) is proposed that corrects this issue by using a proper reference matrix for spherocylindrical cells (Chapter 2). The application of MPCC to experimental spatial distributions of RNA Polymerase and HU is demonstrated. A significantly positive correlation coefficient was obtained for the spatial distribution of the two DNA binding proteins. The MPCC concept could be generalizable to other cell shapes and can be used for both widefield and superresolution images. Translation is one of the most important cellular process. Ribosomes are the central component of translation engaged in protein synthesis from mRNA. Optimal usage of ribosomes is dependent on their spatial organization. In this dissertation, the spatial and temporal organization of translation and a translation elongation protein, Elongation Factor P (EF-P) was investigated in live E. coli cells using superresolution fluorescence microscopy. In rapidly growing E. coli and B. subtilis, strong nucleoid - ribosome segregation is observed. The picture of spatial organization in slowly growing C. crescentus is however completely different. The effect of growth rate on the segregation of ribosomes and nucleoids in E. coli is therefore investigated using two color superresolution fluorescence imaging in Chapter 3. MPCC was used to quantify the degree of segregation between spatial distribution of ribosomes and DNA in E. coli in the two different growth conditions. Slowly growing E. coli showed significant segregation of ribosomes from DNA, unlike slowly growing C. crescentus. Imaged ribosomes were classified as translating and non-translating based on their diffusive trajectories. The fraction of ribosomes engaged in translation in the two growth conditions was quantified. A small but significant reduction of translating ribosomes was observed in slowly growing cells. Simulated spatial distributions in spherocylinders of dimensions same as E. coli that best fit experimental spatial distributions of translating and non-translating ribosomes were used to obtain a cellular map of ribosomes based on their biological functions. Based on the functional map of translating and non-translating ribosomes, it was concluded that a "circulation model" of ribosomes evidently applies to E. coli in all growth conditions. In cells, the translation elongation is discontinuous, often suffering from periods of pauses. The cells have mechanisms to alleviate these pauses, irrespective of the reason for pausing. EF-P is a translation factor that alleviates pausing during translation of polyproline motifs. The spatial distribution and ribosome binding dynamics of EF-P were investigated in Chapter 4 to understand details of mechanism of interaction of EF-P with ribosomes during the translation elongation process. By localizing individual EF-P molecules, the spatial distribution of EF-P was shown to mimic the "three peaked" ribosome distribution in the cells. Photoactivated Localization Microscopy combined with single particle tracking (sptPALM) was employed to distinguish and quantify the different diffusive states of EF-P. The "slow" diffusing EF-P molecules are associated with translating ribosomes whereas the "fast" diffusing ones are EF-P copies searching for a potential binding site on ribosomes. Nearly 30% of EF-Ps are associated with translating ribosomes, implying 1500-6000 EF-P/ribosome complexes in a cell at any given time. This is significantly larger than the estimated 280 pausing events due to polyproline motifs being translated in the cell, suggesting interrogation of ribosomes by EF-P more frequently than the number of pausing events. In addition, faster imaging rates enabled measurement of binding/unbinding timescales of EF-P interactions with ribosomes (Chapter 4). Overall, the reported works explore the synergism of interactions between different components of translation and their spatiotemporal organization. The famous biologist Jacques Monod noted "What was true for the E. coli would be true for the elephant". A better understanding of the intracellular spatial organization of bacterial cells could elucidate the mechanisms that underlie spatial order in higher organisms

Download or read book Pathway Engineering of the Expanding Genetic Code written by John Christopher Anderson and published by . This book was released on 2003 with total page 546 pages. Available in PDF, EPUB and Kindle. Book excerpt: Several projects focused on expanding the genetic code of Escherichia coli to include amino acids beyond the common twenty are described. These include the identification of extended codons, orthogonal pairs, and the engineering of amino acid metabolic pathways in E. coli . To incorporate multiple unnatural amino acids at distinct sites in a polypeptide, multiple noncoding codons are required. To this end efficiently-suppressed 4- and 5-base codons were identified using a selection strategy. To make use of these extended codons, three orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pairs were engineered from archaebacteria. These systems do not cross-react with their E. coli homologs, but efficiently suppress UAG, AGGA, or UGA codons. Using a combination of saturation mutagenesis and genetic selection, several variants of the Pyrococcus horikoshii lysyl-tRNA synthetase were identified that permit the genetic encoding of homoglutamine in response to the codon AGGA in E. coli . In addition to adding new components to the translational machinery of E. coli , we have also added a biosynthetic pathways for p-aminophenylalanine providing the first example of an autonomous 21-amino acid organism that can biosynthesize an unnatural amino acid and incorporate it into protein with no media supplement. Finally, in an effort to incorporate [alpha]-hydroxy acids genetically into proteins we have identified strains deficient in their biotransformation to [alpha]-amino acids in E. coli . In combination with extended codons and orthogonal pairs, these strains set the stage for the evolution of a system that genetically encodes polyesters. The work described here not only allows us to generate proteins and possibly entire organisms with novel properties not restricted by the common twenty amino acids, it also provides an opportunity to explore basic mechanisms of translation.

Download or read book Translation in Mitochondria and Other Organelles written by Anne-Marie Duchêne and published by Springer Science & Business Media. This book was released on 2013-09-13 with total page 264 pages. Available in PDF, EPUB and Kindle. Book excerpt: The present book gives an overview on the similarities and differences of the various translation systems. Moreover, it highlights the mechanisms and control of translation in mitochondria and other organelles such as chloroplasts, plastids and apicoplasts in different organisms. Lastly, it offers an outlook on future developments and applications that might be made possible by a better understanding of translation in mitochondria and other organelles.

Download or read book Spatial Biology of Transcription and Translation in Live E Coli Using Super resolution Fluorescence Microscopy written by and published by . This book was released on 2014 with total page 231 pages. Available in PDF, EPUB and Kindle. Book excerpt: Due to the lack of membrane bound organelles in bacteria, both transcription and translation take place inside the same compartment. The objective of this thesis is to understand the fundamental, as yet unanswered question of where transcription and translation take place inside bacteria. I have focused on the bacterium E. coli, as it is the best characterized organism, both molecularly and physiologically. I have used superresolution microscopy techniques in live E. coli cells to study the spatio-temporal organization of transcription and translation machinery. By localizing individual molecules with 30 nm scale accuracy, I was able to resolve the spatial distribution and dynamics of RNA polymerase (RNAP) and of the ribosomes. The spatial distribution of RNAP mimicked the spatial distribution of the chromosomal DNA, imaged by DNA-stains and superresolution imaging of DNA-binding protein HU. Ribosomes on the other hand were found to be segregated from the chromosomal DNA. By relating the position, dynamics and copy numbers of ribosomes and RNAPs, I was able to conclude that most translation in E. coli is taking place away from the nucleoid in the ribosome-rich regions without transcriptional coupling. This finding is consistent with the timescale of transcription and life time of mRNA. Based on the comparison of spatio-temporal dynamics of chromosomal loci and RNA polymerase, I was able to infer that transcription of protein genes is scattered more or less uniformly throughout the nucleoid. Here the mRNA is translated co-transcriptionally. Once the transcription is terminated, the mRNA diffuses out into the ribosomes-rich regions of the end cap of cytoplasm and is translated until it is degraded by the degradosome machinery. In sharp contrast, transcription of the ribosomal RNA seems to be localized to the periphery of the chromosomal DNA. Such placement of rRNA transcription at the interface between the dense nucleoids and the ribosome-rich regions may be functionally important for efficient ribosome assembly. I also found significant numbers of transcribing RNAPs and translating ribosomes near the cytoplasmic membrane. This provides circumstantial evidence for "transertion hypothesis", which posits the co-transcriptional translation of membrane proteins, which are evidently inserted into the membrane by the Sec-machinery.

Download or read book Cumulated Index Medicus written by and published by . This book was released on 1997 with total page 1860 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Evolution of the Protein Synthesis Machinery and Its Regulation written by Greco Hernández and published by Springer. This book was released on 2016-08-10 with total page 564 pages. Available in PDF, EPUB and Kindle. Book excerpt: The “omics” era has given a new perspective to the findings on the origin and evolution of the process of translation. This book provides insight into the evolution of the translation process and machinery from a modern perspective. Written by leading experts in molecular biology, this text looks into the origins and evolution of the protein synthetic machinery.

Download or read book Studies of Translation in E Coli written by Margareta Faxén and published by . This book was released on 1993 with total page 44 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Gene Expression written by Lynn Cloney and published by . This book was released on 1988 with total page 296 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Dissertation Abstracts International written by and published by . This book was released on 2009 with total page 840 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book A Stoichiometric Model of Escherichia Coli s Macromolecular Synthesis Machinery and Its Integration with Metabolism written by Ines Thiele and published by . This book was released on 2009 with total page 232 pages. Available in PDF, EPUB and Kindle. Book excerpt: Systems biology is a rapidly growing discipline. It is widely believed to have a broad transformative potential on both basic and applied studies in the life sciences. In particular, biochemical network reconstructions are playing a key role as they provide a framework for investigation of the mechanisms underlying the genotype-phenotype relationship. In this thesis, the procedure to reconstruct metabolic networks is illustrated and extended to other cellular processes. In particular, the constraint-based reconstruction and analysis approach was applied to reconstruct the transcriptional and translational (tr/tr) machinery of Escherichia coli. This reconstruction, denoted 'Expression-matrix'/ (E-matrix), represents stoichiometrically all known proteins and RNA species involved in the macromolecular synthesis machinery. It accounts for all biochemical transformations to produce active, functional proteins, tRNAs, and rRNAs known to be involved in macromolecular synthesis in E. coli. An initial study investigated basic properties of the E-matrix, including its capability to produce ribosomes, which was found to be in good agreement with experimental data from literature. Furthermore, quantitative gene expression data could be integrated with, and analyzed in the context of, the resulting constraint-based model. Adding mathematically derived constraints to couple certain reactions in the model allowed the quantitative representation of the size of steady state protein and RNA pools. Furthermore, the E-matrix was integrated with the genome-scale E. coli metabolic model and extended the transcriptional and translational reactions to encompass genes encoding all the respective metabolic enzymes. The resulting Metabolite-Expression-matrix (MExv matrix), has exceeds the predictive capacity of the metabolic model and it can, for example, be used to predict the biomass yield since it represents the production of almost 2,000 proteins. E. coli 's ME-matrix is the first of its kind and represents a milestone in systems biology as demonstrates how to quantitatively integrate 'omics'-datasets into a network context, and thus, to study the mechanistic principles underlying the genotype-phenotype relationship. Possible applications are just beginning to become apparent and may include protein engineering, interpretation of adaptive evolution, and minimal genome design. An integration of the ME-matrix with remaining cellular processes, such as regulation, signaling, and replication, will be a next step to complete the first whole-cell model.