Academic literature on the topic 'Ribosome binding sites'
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Journal articles on the topic "Ribosome binding sites"
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Márquez, V., D. N. Wilson, and K. H. Nierhaus. "Functions and interplay of the tRNA-binding sites of the ribosome." Biochemical Society Transactions 30, no. 2 (April 1, 2002): 133–40. http://dx.doi.org/10.1042/bst0300133.
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The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids. The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e. an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins. Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites. In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals. These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA · EF-Tu · GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA2 · mRNA complex during translocation and the maintenance of the reading frame.
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Dorner, S., and A. Barta. "Probing Ribosome Structure by Europium-Induced RNA Cleavage." Biological Chemistry 380, no. 2 (February 1, 1999): 243–51. http://dx.doi.org/10.1515/bc.1999.032.
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AbstractDivalent metal ions are absolutely required for the structure and catalytic activities of ribosomes. They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metalion induced RNA cleavages are useful for characterising metal ion binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA ofE. coli. The cleavage sites were identified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions. Several Eu3+cleavage sites, mostly those at which a general metal ion binding site had been already identified, were identical to previously described divalent metal ions. Overall, the Eu3+cleavages are most similar to the Ca2+cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.
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Prinz, Anke, Enno Hartmann, and Kai-Uwe Kalies. "Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae." Biological Chemistry 381, no. 9-10 (September 13, 2000): 1025–28. http://dx.doi.org/10.1515/bc.2000.126.
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Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.
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Schaletzky, Julia, and Tom A. Rapoport. "Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane." Molecular Biology of the Cell 17, no. 9 (September 2006): 3860–69. http://dx.doi.org/10.1091/mbc.e06-05-0439.
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We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC–SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC–SRP complexes can overcome the competition by nontranslating ribosomes.
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Krüger, Tim, Hanswalter Zentgraf, and Ulrich Scheer. "Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins." Journal of Cell Biology 177, no. 4 (May 21, 2007): 573–78. http://dx.doi.org/10.1083/jcb.200612048.
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Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC–GC interface.
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Auerbach, Tamar, Inbal Mermershtain, Chen Davidovich, Anat Bashan, Matthew Belousoff, Itai Wekselman, Ella Zimmerman, et al. "The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics." Proceedings of the National Academy of Sciences 107, no. 5 (January 11, 2010): 1983–88. http://dx.doi.org/10.1073/pnas.0914100107.
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Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.
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Lytle, J. Robin, Lily Wu, and Hugh D. Robertson. "The Ribosome Binding Site of Hepatitis C Virus mRNA." Journal of Virology 75, no. 16 (August 15, 2001): 7629–36. http://dx.doi.org/10.1128/jvi.75.16.7629-7636.2001.
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ABSTRACT Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.
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Wittmann-Liebold, Brigitte, Monika Ühlein, Henning Urlaub, Eva-Christina Müller, Albrecht Otto, and Oliver Bischof. "Structural and functional implications in the eubacterial ribosome as revealed by protein–rRNA and antibiotic contact sites." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1187–97. http://dx.doi.org/10.1139/o95-128.
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Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2–iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, andL36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at die peptide level in several proteins that were found to constitute me antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, and L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA–peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 widi a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.Key words: antibiotic-binding site, RNA–peptide-binding sites, protein–RNA interaction in ribosomes, functional role of protein L2.
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Espah Borujeni, Amin, Anirudh S. Channarasappa, and Howard M. Salis. "Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites." Nucleic Acids Research 42, no. 4 (November 14, 2013): 2646–59. http://dx.doi.org/10.1093/nar/gkt1139.
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Abstract The ribosome’s interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long, structured 5′ untranslated regions (5′ UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome’s interactions with structured standby sites, upstream of Shine–Dalgarno sequences, and show that these interactions can modulate translation initiation rates by over 100-fold. We find that an mRNA’s translation initiation rate is controlled by the amount of single-stranded surface area, the partial unfolding of RNA structures to minimize the ribosome’s binding free energy penalty, the absence of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first principles and a four-parameter free energy model to accurately predict the ribosome’s translation initiation rates for 136 synthetic 5′ UTRs with large structures, diverse shapes and multiple standby site modules. The model predicts and experiments confirm that the ribosome can readily bind distant standby site modules that support high translation rates, providing a physical mechanism for observed context effects and long-range post-transcriptional regulation.
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Kalies, K. U., D. Görlich, and T. A. Rapoport. "Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex." Journal of Cell Biology 126, no. 4 (August 15, 1994): 925–34. http://dx.doi.org/10.1083/jcb.126.4.925.
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The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.
Dissertations / Theses on the topic "Ribosome binding sites"
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Berg, Emily Katherine. "Thermodynamics of λ-PCR Primer Design and Effective Ribosome Binding Sites." Thesis, Virginia Tech, 2019. http://hdl.handle.net/10919/89900.
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Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator.
A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression.Master of Science
Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.
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Collins, Paula Grosse. "Ribosome Binding to the Mammalian Endoplasmic Reticulum: A Thesis." eScholarship@UMMS, 1991. https://escholarship.umassmed.edu/gsbs_diss/155.
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Investigators have been attempting to identify the receptor for ribosomes on the rough endoplasmic reticulum (RER) for almost 20 years, yet the ribosome receptor has remained elusive. Rough microsomal membranes contain endogenous ribosomes bound in at least two types of interactions. Loosely associated ribosomes can be removed by extraction with a high ionic strength solution, but ribosomes that were actively engaged in translocation when the membranes were isolated remain tethered to the membrane by a nascent polypeptide (Adelman et al., 1973). The original assay for the ribosome receptor involved stripping all of the endogenous ribosomes off of intact membranes before adding back a quantitated amount of ribosomes. More recent assays have employed detergent solubilization of the membrane and then reconstitution of the membrane proteins into lipid vesicles before adding back ribosomes. In both cases ribosome binding to its receptor is measured in an assay that does not involve translation or translocation.
We utilized a crosslinking assay to attempt to identify membrane proteins that function as a binding site for ribosomes engaged in protein translocation across the endoplasmic reticulum. In vivo bound ribosomes that remain associated with the membrane after extraction with a high ionic strength solution are likely to be bound to a functional translocation site. The water soluble, membrane impermeable, thiol-cleavable crosslinker 3,3'-dithiobis (sulfosuccinimidylpropionate) was selected to limit reaction to protein domains located on the cytoplasmic face of salt extracted microsomal membrane vesicles. A specific subset of RER proteins was reproducibly crosslinked to the endogenous ribosomes. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35 kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180 kD protein that had recently been proposed to be a ribosome receptor (Savitz and Meyer, 1990).
RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes in an in vitro binding assay to determine whether ribosome binding activity could be ascribed to the 180 kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180 kD protein, as determined by Coomassie blue staining of a polyacrylamide gel. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments and functional assays with proteoliposomes demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180 kD protein by chromatography. Taken together, the evidence indicates that the 180 kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.
To continue the investigation of ribosome binding, an assay was designed to characterize ribosome-nascent chain complexes bound to the microsomal membrane during translocation. A series of translocation intermediates consisting of discrete sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. Proteinase K was then used as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport. Only those partially translocated nascent chains of 100 amino acids or less were insensitive to protease digestion by externally added protease. It was concluded that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, shorter nascent polypeptides appear not to have lumenally exposed segments.
Ultimately, a functional assay for the ribosome receptor should include binding studies conducted under physiological conditions. For this purpose, an assay was developed that allowed translation, translocation, and termination of a secretory protein to be monitored with probes designed to independently quantitate translating and non-translating ribosomes. A synchronized wheat-germ translation system was programmed with bovine preprolactin mRNA and aliquots were taken at various time points before and after adding membranes. The samples were then separated into membrane bound and soluble species by centrifugation. RNA was isolated from each supernatant and pellet sample and blotted onto nylon sheets. By probing the dot blots with probes that hybridize with either the 5S RNA of wheat germ ribosomes or the preprolactin transcript, the translating ribosomes could be monitored without the interference of the endogenous canine ribosomes on the membrane. By comparing the total amount of preprolactin transcript that bound to the membrane versus the total amount of wheat germ ribosomes bound to the membrane, it was discovered that the vast majority (>99%) of wheat germ ribosomes that bound to the microsomal membrane were non-translating ribosomes. In later experiments it was found that the non-translating ribosomes did not compete with the translating ribosomes; under all conditions tested, the translating ribosomes had access to translocation sites on the microsomal membrane. One interpretation of this data is that all ribosome binding sites are not identical. It may be that functional sites for translocation are a distinct subclass of total ribosome binding sites. Another interpretation is that a ribosome in a nascent chain-SRP complex has a much higher affinity for the ribosome receptor than nontranslating ribosomes or 60S subunits. Perhaps the non-translating ribosome can not compete with ribosomes engaged in translocation. As stated earlier, ribosomes do make at least two kinds of interactions with the microsomal membrane surface. This data may be indicative of those types of interactions.
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Tuck, Laura. "Structural and synthetic biology study of bacterial microcompartments." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33180.
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Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme's substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels.
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Kaminishi, Tatsuya, Andreas Schedlbauer, Attilio Fabbretti, Letizia Brandi, Lizarralde Borja Ochoa, Cheng-Guang He, Pohl Milon, Sean R. Connell, Claudio O. Gualerzi, and Paola Fucini. "Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A." Oxford University Press, 2015. http://hdl.handle.net/10757/608247.
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Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding.
Revisión por pares
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Phelps, Steven Scott. "tRNA interactions in the ribosomal A-site that are important for binding, decoding, and translocation /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2003. http://wwwlib.umi.com/cr/ucsd/fullcit?p3112867.
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Mao, Hongyuan 1969. "Structure determination of a yeast ribosomal protein L30 and pre-mRNA binding site complex by NMR spectroscopy." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/49674.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1998.Includes bibliographical references (p. 342-353).
The yeast (Saccharomyces cerevisiae) ribosomal protein L30 and its auto-regulatory pre-mRNA binding site provide one of the best examples the critical role of protein-RNA interactions in regulation of RNA processing and control of gene translation. A model system for this interaction, which includes the ribosomal L30 protein and the phylogenetically conserved RNA segment for auto-regulation, was studied using nuclear magnetic resonance (NMR) spectroscopy. The L30 protein recognizes and binds tightly to the stem-internal loop-stem RNA, the recognition elements of which lie mostly on the conserved two-plus-five asymmetric purine-rich internal loop. NMR characterizations were carried out on both the free and bound forms of the protein and the RNA. Detailed analyses of the protein revealed that the main architecture, a fourstranded n-sheet sandwiched between four a-helices, is present both in the free and in the bound form. There are however, substantial local perturbations that accompany RNA binding, the largest of which have been mapped onto the loops connecting Strand A and Helix 2, Strand B and Helix 3, Helix 4 and Strand D. In contrast to the protein, the internal loop of the RNA undergoes significant changes upon complex formation, and the most distinct observation was the formation of the G 11G56 reverse Hoogsteen mismatch pair. Structure modeling using simulated annealing in restrained molecular dynamics was carried out in X-PLOR. Detailed analyses of the complex structure reveal that the protein recognizes the RNA mostly along one side of the internal loop with five purines. The interactions are divided further into two sections. One region consists of mostly aromatic stacking and hydrophobic contacts from Leu25, Phe85 and Val87 of the protein to G56 of the RNA. The other region consists of mostly specific contacts, which include recognition of A57 by Asn 48, and G58 by Arg 52. The L30 protein- RNA complex structure thus determined using NMR spectroscopy not only provides a detailed insight for understanding the structure-function relationship regarding the yeast auto-regulation, it also further demonstrates the important role of the protein-RNA interaction in controlling RNA processing and gene translation.
by Hongyuan Mao.
Ph.D.
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Yang, Grace. "Application of the Adaptive Poisson Boltzmann Solver on the investigation of the small oligonucleotide A-site model and 30S ribosomal subunit binding to aminoglycosidic antibiotics /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3170239.
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Toddo, Stephen. "Engineering membrane proteins for production and topology." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-116598.
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The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.
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Bandmann, Nina. "Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification." Doctoral thesis, Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4318.
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Tang, Shiyuyun. "Improving algorithms of gene prediction in prokaryotic genomes, metagenomes, and eukaryotic transcriptomes." Diss., Georgia Institute of Technology, 2016. http://hdl.handle.net/1853/54998.
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Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack.
In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools.
Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods.
Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.
Book chapters on the topic "Ribosome binding sites"
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Wower, Jacek, Lee A. Sylvers, Kirill V. Rosen, Stephen S. Hixson, and Robert A. Zimmermann. "A Model of the tRNA Binding Sites on the Escherichia Coli Ribosome." In The Translational Apparatus, 455–64. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4615-2407-6_43.
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Mazen, Alice, Daniel Lamarre, Guy Poirier, Gérard Gradwohl, and Gilbert de Murcia. "Localization of the Zinc-Binding Sites in the DNA-Binding Domain of the Bovine Poly(ADP-Ribose) Polymerase." In ADP-Ribose Transfer Reactions, 89–93. New York, NY: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4615-8507-7_17.
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Ballesta, Juan P. G. "The Structure of the Antibiotic Binding Sites in Bacterial Ribosomes." In The Translational Apparatus of Photosynthetic Organelles, 179–95. Berlin, Heidelberg: Springer Berlin Heidelberg, 1991. http://dx.doi.org/10.1007/978-3-642-75145-5_15.
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Rheinberger, H. J., A. Gnirke, H. Saruyama, P. Wurmbach, and K. H. Nierhaus. "Three Ribosomal tRNA-Binding Sites Involved in the Elongation Process." In Gene Manipulation and Expression, 455–77. Dordrecht: Springer Netherlands, 1985. http://dx.doi.org/10.1007/978-94-011-6565-5_33.
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Nierhaus, K. H., H. J. Rheinberger, U. Geigenmüller, A. Gnirke, H. Saruyama, S. Schilling, and P. Wurmbach. "Three tRNA Binding Sites Involved in the Ribosomal Elongation Cycle." In Springer Series in Molecular Biology, 454–72. New York, NY: Springer New York, 1986. http://dx.doi.org/10.1007/978-1-4612-4884-2_26.
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Rodnina, Marina V., Rainer Fricke, and Wolfgang Wintermeyer. "Kinetic Fluorescence Study on EF-Tu-Dependent Binding of Phe-tRNAPhe to the Ribosomal a Site." In The Translational Apparatus, 317–26. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4615-2407-6_30.
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Liljas, A. "Ribosome Binding Site." In Encyclopedia of Genetics, 1723. Elsevier, 2001. http://dx.doi.org/10.1006/rwgn.2001.1127.
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Liljas, A. "Ribosome Binding Site." In Brenner's Encyclopedia of Genetics, 247. Elsevier, 2013. http://dx.doi.org/10.1016/b978-0-12-374984-0.01338-3.
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Salis, Howard M. "The Ribosome Binding Site Calculator." In Methods in Enzymology, 19–42. Elsevier, 2011. http://dx.doi.org/10.1016/b978-0-12-385120-8.00002-4.
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Ofengand, James, Robert Denman, Kelvin Nurse, Arnold Liebman, David Malarek, Antonino Focella, and Gladys Zenchoff. "[25] Affinity labeling of tRNA-binding sites on ribosomes." In Methods in Enzymology, 372–97. Elsevier, 1988. http://dx.doi.org/10.1016/s0076-6879(88)64056-0.
Full textConference papers on the topic "Ribosome binding sites"
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Małkiewicz, A. J., M. Marszałek, A. B. Miśkiewicz, E. Sochacka, R. Guenther, and P. F. Agris. "Site-specifically modified sequences of human tRNA3Lys and E. coli tRNALys anticodon arms: synthesis and binding to ribosome." In XIth Symposium on Chemistry of Nucleic Acid Components. Prague: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 1999. http://dx.doi.org/10.1135/css199902328.
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Hu, Jun, and Jing Zhang. "Co-occurrence of core of binding sites for transcription factors in intronic region of Saccharomyces cerevisiae ribosomal protein genes." In 2010 International Conference on Bioinformatics and Biomedical Technology. IEEE, 2010. http://dx.doi.org/10.1109/icbbt.2010.5479005.
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