Mutation and Modulation of Translational Machinery in E. Coli PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Mutation and Modulation of Translational Machinery in E. Coli PDF full book. Access full book title Mutation and Modulation of Translational Machinery in E. Coli by Corey M. Dambacher. Download full books in PDF and EPUB format.
Author: Corey M. Dambacher
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 366
Get Book Here
Book Description
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.
Author: Corey M. Dambacher
Publisher:
ISBN:
Category : Escherichia coli
Languages : en
Pages : 366
Get Book Here
Book Description
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.
Author: Piotr Lagod
Publisher:
ISBN:
Category :
Languages : en
Pages : 68
Get Book Here
Book Description
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.
Author: Assaf Katz
Publisher: Frontiers Media SA
ISBN: 2889663604
Category : Science
Languages : en
Pages : 137
Get Book Here
Book Description
Author: Kaj Bohman
Publisher:
ISBN: 9789155415594
Category :
Languages : en
Pages : 29
Get Book Here
Book Description
Author: Charles T. Backus
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
Author: Sonisilpa Mohapatra
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
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
Author: John Christopher Anderson
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages : 546
Get Book Here
Book Description
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.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 231
Get Book Here
Book Description
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.
Author: Anne-Marie Duchêne
Publisher: Springer Science & Business Media
ISBN: 3642394264
Category : Science
Languages : en
Pages : 264
Get Book Here
Book Description
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.
Author: Greco Hernández
Publisher: Springer
ISBN: 3319394681
Category : Science
Languages : en
Pages : 564
Get Book Here
Book Description
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.
