A new technique developed at UC Davis may have broken the barrier to rapid assembly of pure protein synthesis machinery outside of living cells.
The overall aim of this thesis was to investigate how genome engineering might be used to generate Escherichia coli strains with increased capacities for recombinant protein production. As translation constitute a possible bottleneck in recombinant production processes [Mahalik et. al, 2014] this work focused on evaluating and increasing translational capacities in E. coli. Two methods were used to evaluate translational capacities in E. coli: 1. Two plasmid reporter systems were established to investigate levels of ribosome expression. These plasmids carried red fluorescent protein genes (mCherry), under control of ribosomal promoters. Levels of expression of ribosomal constituents were evaluated by measuring fluorescence from strains carrying reporter plasmids.. 2. Exponential phase growth rates were used to assess translational capacities. Protein synthesis is generally the growth rate limiting factor during exponential phases, and a positive linear correlation between growth rates and ...
Upon EV-A71 infection of a host cell, EV-A71 RNA is translated into a viral polyprotein. Although EV-A71 can use the cellular translation machinery to produce viral proteins, unlike cellular translation, which is cap-dependent, the viral RNA genome of EV-A71 does not contain a 5′ cap and the translation of EV-A71 protein is cap-independent, which is mediated by the internal ribosomal entry site (IRES) located in the 5′ UTR of EV-A71 mRNA. Like many other eukaryotic viruses, EV-A71 manipulates the host cell translation devices, using an elegant RNA-centric strategy in infected cells. During viral translation, viral RNA plays an important role in controlling the stage of protein synthesis. In addition, due to the cellular defense mechanism, viral replication is limited by down-regulating translation. EV-A71 also utilizes protein factors in the host to overcome antiviral responses or even use them to promote viral translation rather than host cell translation. In this review, we provide an introduction
Abstract: The development of cancer is a consequence of mutations that lead to dysfunctional cell processes such as unrestrained cell proliferation resistance to apoptosis and improper regulation of cell processes such as translation. Cell proliferation and apoptosis are linked to specific gene expression events regulated by protein synthesis which begins with the binding of various eukaryotic initiation factors (eIF) to mRNA and ribosomes to initiate translation. eIF4G-1 catalyzes two types of translation initiation. Cap-dependent translation requires eIF4E to bind a 5-methylated mRNA cap and eIF4G-1. This in turn facilitates recruitment and promotes translation of cell cycle and growth-related proteins. Cap-independent translation initiates internally through internal ribosome entry sites (IRES) in the 5 UTR of mRNA and promotes translation of apoptotic mRNAs such as Apaf-1. Previous studies found that eight variants of eIF4G-1 mRNA exist and five protein isoforms can be resolved by ...
There is extensive evidence that the structurally complex, negatively charged GAG side chains of HSPGs are crucial for signaling by diverse secreted ligands, including BMPs. Although the distribution of HSPGs is generally assumed to be ubiquitous, our data showing that GAGs are absent in the first three hours after egg laying but are rapidly synthesized thereafter demonstrates that HSPG expression can, in fact, be precisely temporally regulated. We have shown that this regulation results from an absence of enzymes essential for HSPG synthesis, owing to a block to their translation. Furthermore, our data strongly support the notion that the translational control mechanism involves the use of developmentally regulated internal ribosome entry sites (IRESs) in mRNA 5′ UTRs. Translationally blocking GAG synthesis may enable generation of the Dpp/Scw activity gradient in the absence of GAGs, while permitting rapid GAG synthesis from the abundant maternal mRNA supply coincident with the expression of ...
Aims: Rational use of antibiotics against the betacoronavirus SARS-CoV-2 responsible for the COVID-19 pandemic. Objective: Repositioning and repurposing adequate antibiotics to cure the Coronavirus Disease 2019 (COVID-19). Background: It is widely accepted that viral infections such as the SARS-CoV-2 cannot be cured by antibiotics, whereas bacterial infections can. It is because the SARS-CoV-2 virus has no protein synthesis machinery (usually targeted by antibiotics) to produce from its RNA genome, the viral proteins and enzymes essential for its replication and/or for the assembly of viral particles. However, the antibiotics must be capable of inhibiting the ribosomes of the protein synthesis machinery of the SARS-CoV-2-infected human host cells, in order to prevent them from synthesizing new proteins that they do not need, but are needed for the virus to spread. Unfortunately, the only antibiotic capable of selectively inhibiting the human 80S ribosomes, namely cycloheximide, was found to
Deregulation of translational control can promote cellular transformation. Protein synthesis and the expression of components of the translation machinery are elevated in cancers and contribute to tumorigenesis (1-5, 7). Here, we show that nuclear ErbB2 promotes binding of RNA Pol I to rDNA, co-occupies the rRNA gene with β-actin and RNA Pol I, and stimulates rRNA production and protein translation independently of traditional ErbB2 downstream PI3-K and ERK signalings, suggesting that nuclear ErbB2 may contribute to oncogenesis by upregulating total cellular translation. rRNA synthesis by RNA Pol I plays a critical role in production of mature ribosomes that are central protein synthesis machinery of the cells. Perturbation of RNA Pol I activity as well as rRNA and protein biosynthesis (i.e., translation control) by oncoproteins such as Myc or tumor suppressors p53, RB, and ADP ribosylation factor has been reported to be associated with tumor development (1, 2, 7-10). The capability of Myc to ...
Localization of the cytoplasmic translation and mitochondrial import machinery relative to newly-made mitochondrial transcripts. Cells were grown in BrU for 30
TY - JOUR. T1 - Electrically stimulated contraction accelerates protein synthesis rates in adult feline cardiocytes. AU - Ivester, C. T.. AU - Kent, R. L.. AU - Tagawa, H.. AU - Tsutsui, Hiroyuki. AU - Imamura, T.. AU - Cooper IV, G.. AU - McDermott, P. J.. PY - 1993/1/1. Y1 - 1993/1/1. N2 - Cardiocytes were induced to contract via electrical field stimulation with an 8 V/cm electrical square-wave pulse of 5 ms at 0.125-2.0 Hz for up to 6 h. Protein synthesis rates were measured as rate of incorporation of [3H]- phenylalanine into total cell protein. Rates of protein synthesis were accelerated 43 ± 4%, P , 0.001, by 4 h. The acceleration of total protein synthesis showed a frequency dependence between 0.125 and 0.5 Hz. In addition to accelerating rates of total protein synthesis, electrical stimulation of contraction accelerated fractional rates of synthesis of myosin heavy chain by 42 ± 8%, P , 0.05. Protein synthesis rates were not accelerated upon electrical stimulation using subthreshold ...
Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential ‘scanning’ of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the
A technique for image alignment with global translation and linear stretch determines translation parameters for three corresponding linearly displaced blocks in a reference image and a corresponding distorted test image. From the differences between the translation parameters for the three blocks the presence of stretch is detected and, if detected, a stretch factor is estimated. The estimated stretch factor is used as a starting point to stretch the reference image to overlap the distorted test image as a refinement process. The resulting refined stretch factor is then used in a reverse stretch process to shrink the distorted test image, and the distorted test image is then aligned with the reference image to obtain picture quality metrics.
Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA
Quiescence (G0) represents an assortment of reversible, cell cycle-arrested states that are resistant to unfavorable conditions and associated with cancer persistence. G0 involves regulated gene expression with selective mRNA expression and decreased canonical translation. Low mTOR activity in G0 activates the cap complex inhibitor, eIF4EBP, and impairs canonical translation. The alternative translation mechanisms in G0 remain to be uncovered. Our data show that microRNAs, regulatory, non-coding RNAs that target distinct mRNAs to alter gene expression, can associate with alternative complexes and translation factors to regulate specific mRNA translation in G0. One subset of transcripts expressed in G0 includes specific mRNAs recruited by an FXR1a-associated microRNP (microRNA-protein complex) for translation activation in G0 mammalian cells. MicroRNPs predominantly mediate repression and downregulation; however, FXR1a-microRNP lacks conventional microRNP repressors, and instead, contains a ...
TY - JOUR. T1 - Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote. AU - Miyagishima, Shin Ya. AU - Fujiwara, Takayuki. AU - Sumiya, Nobuko. AU - Hirooka, Shunsuke. AU - Nakano, Akihiko. AU - Kabeya, Yukihiro. AU - Nakamura, Mami. N1 - Funding Information: We thank A. Yamashita for technical support and K. Fukaya and BSIs Research Resources Center of RIKEN for LC-MS/MS analyses. This study was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (no. 19870033 to S.-y.M.) and by Core Research for Evolutional Science and Technology (CREST) Program of Japan Science and Technology Agency (JST) (to S.-y.M.).. PY - 2014/5/8. Y1 - 2014/5/8. N2 - Circadian rhythms of cell division have been observed in several lineages of eukaryotes, especially photosynthetic unicellular eukaryotes. However, the mechanism underlying the circadian regulation of the cell cycle and the nature of the advantage conferred remain ...
The unfolded protein response (UPR) is an intracellular signaling pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER). The UPR results in an increase in transcription of ER-resident proteins that facilitate protein folding in the ER. A key regulatory step in UPR activation is the regulated splicing of HAC1 mRNA, which encodes Hac1p, a transcription factor dedicated to this pathway. Hac1p can be detected only when the spliced form of HAC1 mRNA (termed HAC1i mRNA, for induced) is produced; this was surprising because the unspliced HAC1u mRNA (HAC1u for uninduced) is equally stable in cells.. ...
A translation lookaside buffer (TLB) stores translation entries. The translation entries include a virtual address, a physical address and a memory local/not-local flag. When a processor is in a low power/local memory mode a virtual address is received. A matching translation entry has a local/not-local flag. Upon the local/not-local flag indicating the physical address of the matching translation entry being outside the local memory, an out-of-access-range memory access exception is generated.
Learn about DNA Delivery and Protein Synthesis. Topics cover DNA delivery, screening of recombinant clones, protein synthesis machinery, and expression systems.
Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, ...
Every protein in a cell is produced from a transiently synthesized messenger molecule, termed mRNA. This mRNA is then recognized by a cellular machinery that translates the base sequence of mRNA into its corresponding protein (which is a sequence of amino acids). This protein synthesis machinery, termed ribosome, is actually a ribozyme, i.e. it is a catalytically active assembly of several RNA molecules. Consequently, RNAs do not only serve as messenger molecules or perform structural functions as e.g. in tRNA but may also act as catalysts that perform biochemical reactions as is the case for protein enzymes. Of course, the ribosome also contains numerous proteins as it is a very complex ribonucleoprotein particle but these predominantly serve structural functions, e.g. to give the ribosome its shape. Fascinatingly, the initial analysis of the human genome sequence revealed that, apparently, only a very small fraction of the DNA of our genome is encoding proteins. Scientists at first thought ...
To gain preferential access to the protein synthesis machinery and to disrupt induction of antiviral responses by infected cell many viruses block host gene expression. This blockade is called host shutoff and it is mediated by viral factors that either destroy host messenger RNAs (mRNAs) or interfere with their synthesis. Influenza A virus (IAV) encodes…
Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo . The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from ...
Eukaryotic cells respond to unfolded proteins in their endoplasmic reticulum (ER stress), amino acid starvation, or oxidants by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). This adaptation inhibits general protein synthesis while promoting translation and express …
By using a systemic mtEF4 gene knockout mouse model, researchers from the Institute of Biophysics of the Chinese Academy of Sciences found that mtEF4 knockout damages the oxidative phosphorylation function in germ cells of male mice, thus causing male sterility.
In mammals, such as humans, DNA contains genetic instructions that are transcribed-or copied-into RNA. While DNA remains in the cells nucleus, RNA carries the copies of genetic information to the rest of the cell by way of various combinations of amino acids, which it delivers to ribosomes. The ribosomes link the amino acids together to form proteins that then carry out functions within the human body. The viral RNA is sneaky: its features cause the protein synthesis machinery of our cells to mistake it for RNA produced by our own DNA. COVID-19 enters the body through the nose, mouth, or eyes and attaches to our cells. Once the virus is inside our cells, it releases its RNA. Our hijacked cells serve as virus factories, reading the viruss RNA and making long viral proteins to compromise the immune system. The virus assembles new copies of itself and spreads to more parts of the body and-by way of saliva, sweat, and other bodily fluids-to other humans. RNA research at the University of Rochester
HIV causes the serious disease AIDS yet it has a fascinating life cycle that can be used to teach many key concepts in molecular biology. With this model your students will see how the virus uses parts of the infected cells protein synthesis machinery to replicate itself. Using real DNA sequence, the crucial steps of r
Organisms use highly accurate molecular processes to transcribe their genes and a variety of mRNA quality control and ribosome proofreading mechanisms to maintain intact the fidelity of genetic information flow. Despite this, low level gene translational errors induced by mutations and environmental factors cause neurodegeneration and premature death in mice and mitochondrial disorders in humans. Paradoxically, such errors can generate advantageous phenotypic diversity in fungi and bacteria through poorly understood molecular processes. In order to clarify the biological relevance of gene translational errors we have engineered codon misreading in yeast and used profiling of total and polysome-associated mRNAs, molecular and biochemical tools to characterize the recombinant cells. We demonstrate here that gene translational errors, which have negligible impact on yeast growth rate down-regulate protein synthesis, activate the unfolded protein response and environmental stress response pathways, and down
Figure 2. The phenotype of FoxC1 depleted gastulae. A:Xenopus FoxC1 pseudoalleles aligned against the reverse complement of the morpholino oligo used in the study using Clustal alignment in Macvector. B: FoxC1 MO (MO) blocks translation of FoxC1 mRNA (FoxC1) in a dose-responsive fashion, but does not block translation of a protected FoxC1 RNA, (MOR-), in an in vitro translation assay. C: HA-tagged Fox1 mRNA was injected alone or together with different doses of FoxC1 MO at the 2-cell stage to confirm a reduction of FoxC1 translation. D-G: Wild type sibling embryos and embryos injected with 40 ng MO (MO), or 40 ng MO and 50 pg MO-R FoxC1 mRNA (MO+RNA) were cultured until early (stage 10), mid (stage 11), and late gastrula (stage 11.5) stages before freezing and analyzing by real time RT-PCR for the expression of Mespo (D), Endodermin (E), Bix4 (F), and CK1ϵ (G). Expression levels are normalized to ODC. H,I: Whole mount in situ hybridization on FoxC1-depleted mid-gastrulae (bottom) as compared to ...
Abstract : Escherichia coli holds important secrets of the life, and persists with its metallic sheen among the model organisms used in modern biology research. Exploration of core biological processes in this classical model organism continues to provide a wealth of new information relevant to all life forms. In my lecture, I will discuss how the excellent amenability of E. coli to the classical genetics approaches in conjunction with the modern molecular methods continues to unravel evolutionary complexities of biological systems. In particular, I will discuss examples of how E. coli has been instrumental in addressing many questions in our pursuit of understanding the protein synthesis machinery.. ...
If you have a question about this talk, please contact Florian Markowetz.. Translation efficiency is determined, in part, by the supply-to-demand relationships between the tRNA and mRNA pools in cells. To better understand translation we perturb the system through tRNA gene manipulation and recoding open reading frames. We then follow such perturbed and genome engineered bacteria and yeast cells through lab evolution and comparative genomics and reveal how evolution acts to adapt supply to demand. I will also discuss our study of the cancerous tRNA and mRNA pools. I will show how cancers sustain high translation efficiency of genes that carry out cell autonomous, hence oncogenic, functionalities.. Hosted by Duncan Odom. This talk is part of the Seminars on Quantitative Biology @ CRUK Cambridge Institute series.. ...
Large and systematic mRNA library design disentangles the complex sequence determinants of translation efficiency in bacteria. Comparative analyses of natural and mutated sequences have been used to probe mechanisms of gene expression, but small sample sizes may produce biased outcomes. We applied an unbiased design-of-experiments approach to disentangle factors suspected to affect translation efficiency in E. coli. We precisely designed 244,000 DNA sequences implementing 56 replicates of a full factorial design to evaluate nucleotide, secondary structure, codon and amino acid properties in combination. For each sequence, we measured reporter transcript abundance and decay, polysome profiles, protein production and growth rates. Associations between designed sequences properties and these consequent phenotypes were dominated by secondary structures and their interactions within transcripts. We confirmed that transcript structure generally limits translation initiation and demonstrated its physiological
Plants respond to changes in sugar concentrations by altering their transcriptional profile and their metabolic processes. Sucrose triggers the translational repression of the transcription factor bZIP11 in Arabidopsis thaliana. Rahmani et al. show that this repression requires the 5′-leader sequence of bZIP11, which, when transferred to a reporter gene, decreased the expression of the reporter gene product (luciferase) in response to sucrose, consistent with sugar-induced translational repression. Deletion analysis revealed that the second upstream open reading frame (uORF2) was required. When the mRNA sequence of uORF2 was mutated without affecting the encoded peptide, sugar-induced translational repression occurred. Transplantation of the 82 nucleotides of the uORF2 sequence into the promoter of a gene not normally regulated by sucrose caused the production of the reporter gene to decrease in response to sucrose. Mutation of the encoded peptide, or changing the length of the encoded ...
FIGURE 1. Posttranscriptional and translational mechanisms of gene regulation in CD4+ T cell subsets. (1) Increased translational events cause differentiated Tregs to proliferate, whereas Foxp3 induction and Treg differentiation are facilitated by decreased translation and posttranscriptional mechanisms. (2) miR-155 can cause an increase in IFN-Rα expression and initiate naive T cell differentiation toward the Th1 cell type. (3) miR-155 also blocks SOCS1 translation and, in turn, induces Foxp3 transcription. (4) Environmental cues like hypoxia can influence T cell differentiation into various lineages, with the Th17 and Treg types shown in this figure. (5) miR-126 blocks the translation of PU.1, an inhibitor of GATA-3 interaction with DNA, thus promoting the development of Th2 cells. (6) Interaction of eIF4E with mature mRNAs governs translation, and its activity is controlled by the eIF4E-binding proteins. (7) Poly(A)-binding proteins cause circularization of mRNAs and increase translation. ...
A rapid method for gene expression analysis, PURExpress® is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation. The relative nuclease-free and protease-free nature of the PURExpress platform preserves the integrity of DNA and RNA templates/ complexes and results in proteins that are free of modification and degradation. Transcription and translation are carried out in a one-step reaction, and require the mixing of only two tubes. With results available in a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies.
View Notes - Control of Gene Expression from BSC BSC1005 at Broward College. mRNA and translational mechanisms control the synthesis of protein after mRNA has been produced. Operons Operons are
The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5 end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3 to 5 mRNA degradation activity is ...
Protein synthesis is the ultimate step of gene expression and a key control point for regulation. In particular, it enables cells to rapidly manipulate protein production without new mRNA synthesis, processing, or export. Recent studies have enhanced our understanding of the translation initiation p …
p>The checksum is a form of redundancy check that is calculated from the sequence. It is useful for tracking sequence updates.,/p> ,p>It should be noted that while, in theory, two different sequences could have the same checksum value, the likelihood that this would happen is extremely low.,/p> ,p>However UniProtKB may contain entries with identical sequences in case of multiple genes (paralogs).,/p> ,p>The checksum is computed as the sequence 64-bit Cyclic Redundancy Check value (CRC64) using the generator polynomial: x,sup>64,/sup> + x,sup>4,/sup> + x,sup>3,/sup> + x + 1. The algorithm is described in the ISO 3309 standard. ,/p> ,p class=publication>Press W.H., Flannery B.P., Teukolsky S.A. and Vetterling W.T.,br /> ,strong>Cyclic redundancy and other checksums,/strong>,br /> ,a href=http://www.nrbook.com/b/bookcpdf.php>Numerical recipes in C 2nd ed., pp896-902, Cambridge University Press (1993),/a>),/p> Checksum:i ...
Accumulation of viral products such as RNA replication intermediates and viral proteins represents a potential stressor for host cells. Rapidly after detection, host cells respond by implementing multiple appropriated defense mechanisms, including innate immune and stress responses. The strongest response to several forms of stress, including viral infections, is a global reduction of protein synthesis which promotes cellular survival. Translation suppression is induced by the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor-2 (eIF2α), thereby causing stalling of translation initiation and accumulation of stalled pre-initiation complexes in cytosolic stress granules (SGs). Viruses do not package ribosomes and therefore fully rely on the utilization of the host translation machinery to ensure viral protein synthesis, replication and virus progeny production. As a consequence, virus survival depends on the establishment of a delicate and fine-tuned balance ...
The DNA that makes up an organisms genome contains genes that hold the nucleic acid codes for protein synthesis, resulting in the production of unique proteins that perform numerous and specific functions. Protein synthesis involves two steps: transcription and translation. Transcription creates a complementary RNA copy of a DNA sequence and translation is the subsequent process where RNA is used to synthesize the actual protein from amino acids. Inhibition of this translation step has the effect of blocking protein production and ultimately its function. Sigma-Aldrich offers several small molecules that inhibit protein translation, making these compounds useful research tools to better understand the role proteins play in certain disease states.
By by ... Detlef --- dlls/mlang/tests/mlang.c , 7 +++++-- 1 files changed, 5 insertions(+), 2 deletions(-) diff --git a/dlls/mlang/tests/mlang.c b/dlls/mlang/tests/mlang.c index fb37e27..0da31b5 100644 --- a/dlls/mlang/tests/mlang.c +++ b/dlls/mlang/tests/mlang.c @@ -121,6 +121,9 @@ static const WCHAR de_engb2[] ={E,n,g,l,i,s,c,h, , K,0xF6,n,i,g,r,e,i,c,0}; static const WCHAR de_enus[] = {E,n,g,l,i,s,c,h, , (,U,S,A,),0}; +static const WCHAR de_enus2[] ={E,n,g,l,i,s,c,h, , + (,V,e,r,e,i,n,i,g,t,e, , + S,t,a,a,t,e,n,),0}; static const WCHAR de_de[] = {D,e,u,t,s,c,h, , (,D,e,u,t,s,c,h,l,a,n,d,),0}; static const WCHAR de_deat[] = {D,e,u,t,s,c,h, , @@ -184,11 +187,11 @@ static const info_table_entry info_table[] = { {MAKELANGID(LANG_ENGLISH, SUBLANG_NEUTRAL), MAKELANGID(LANG_GERMAN, SUBLANG_DEFAULT), TODO_NAME, en, de_en}, ...
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Studies like this found that 20 to 40 grams of protein stimulates maximal protein synthesis.. That is, 20 to 40 grams of protein in a meal is as anabolic as you can get and increasing intake beyond this amount accomplishes little-to-nothing in terms of muscle/tissue growth and repair.. This limit to protein synthesis is then construed as a limit to absorption.. If eating more protein doesnt further elevate protein synthesis rates, the story goes, that must mean your body cant handle any more, right?. Wrong.. How high protein synthesis rates go is only one dimension of what happens with them when you eat. How long they remains elevated is equally if not more important.. For example, research shows that 30 grams of whey protein spikes protein synthesis rates higher than 30 grams of casein does. But, due to wheys rapid absorption, protein synthesis rates also fall back to baseline sooner.. (Whey causes a shorter, larger increase in protein synthesis whereas casein causes a smaller, longer ...
It sounds like the autoradiography is done directly on the gel. Since the protein contains the hot sulfur, you dont need a radiolabeled antibody to detect it. You can infer the mass of the protein from its mobility (location of gel band). However, if you need to unambiguously prove the identity of the protein you might need a Western as well, since your radiolabelled gel gives mass information but not an epitope-antigen interaction. If you have a single mRNA produced in the transcription-translation system then that simplifies the system. Without knowing the source of the DNA and how many different kinds of mRNA would be expected to be present, its tough to tell whether a Western would be necessary to prove your band is what it should be ...
The rbcL 5′-UTR contains an SDsequence at the prokaryotic consensus position, whereas theatpB 5′-UTR lacks one. Therefore, it is likely that theatpB 5′-UTR has mechanisms other thanSD-ASD interactions in the 5′-UTR to facilitate translation initiation. The mechanism of translational activation is not known. A candidate for translational activation of theatpB mRNA is the general S1 ribosomal protein mediated mechanism with affinity for AU-reach sequences in the 5′-UTR (Franzetti et al., 1992; Alexander et al., 1998). A specific control factor could be encoded in a maize atp1 gene homolog (McCormac and Barkan, 1999). Our mutagenesis study indicates that the role of sequences downstream of AUG in plastid translation is highly dependent on the individual mRNA. The consequence of mutagenesis onrbcL translation was a dramatic 35-fold drop in NPTII levels from 11% (w/w) to 0.3% (w/w). It is apparent that translation of the chimeric mRNA is highly dependent on sequences directly downstream of ...
Research in this section is focused on understanding translational regulatory mechanisms and the molecular details of protein synthesis in eukaryotic cells. Given its critical importance as the ultimate step in gene expression and its significant energy r
P.341 left column: [Investigators] show that cecER and the pmaER (cytoplasmic face) have the highest ribosome densities ranging from ∼600 to 1,100 ribosomes/µm^2 for the cecER and ∼550 to 900 ribosomes/µm^2 for the pmaER (cytoplasmic face). The ribosome density of yeast cecER is similar to that of mitotic mammalian BSC1 cell cisternae, which was determined by similar methods (1,000 ± 300 µm^2, primary source). The tubER is bound by ribosomes, although it does have less bound ribosomes than the other domains (typically ∼250-400 ribosomes/µm^2 density for tubER, Fig. 5 E). ER ribosome densities are generally lower in the bud than in the mother, suggesting that ribosomes may dissociate and then need to reassociate during inheritance (compare densities in Fig. 5, E and F). Together, these data demonstrate that tubER does have less bound ribosomes than cecER and pmaER. However, membrane curvature alone does not define ER ribosome density because pmaER and cecER have very similar levels of ...
Hepatitis C virus (HCV) protein synthesis is mediated by a highly conserved internal ribosome entry site (IRES), mostly located at the 5′ untranslatable region (UTR) of the viral genome. The translation mechanism is different from that used by cellular cap-mRNAs, making IRESs an attractive target site for new antiviral drugs. The present work characterizes a chimeric RNA molecule (HH363-50) composed of two inhibitors: a hammerhead ribozyme targeting position 363 of the HCV genome and an aptamer directed towards the essential stem-loop structure in domain IV of the IRES region (which contains the translation start codon). The inhibitor RNA interferes with the formation of a translationally active complex, stalling its progression at the level of 80S particle formation. This action is likely related to the effective and specific blocking of HCV IRES-dependent translation achieved in Huh-7 cells. The inhibitor HH363-50 also reduces HCV RNA levels in a subgenomic replicon system. The present findings
Usage of presumed 5′UTR or downstream in-frame AUG codons, next to non-AUG codons as translation start codons contributes to the diversity of a proteome as protein isoforms harboring different N-terminal extensions or truncations can serve different functions. Recent ribosome profiling data revealed a highly underestimated occurrence of database nonannotated, and thus alternative translation initiation sites (aTIS), at the mRNA level. N-terminomics data in addition showed that in higher eukaryotes around 20% of all identified protein N termini point to such aTIS, to incorrect assignments of the translation start codon, translation initiation at near-cognate start codons, or to alternative splicing. We here report on more than 1700 unique alternative protein N termini identified at the proteome level in human and murine cellular proteomes. Customized databases, created using the translation initiation mapping obtained from ribosome profiling data, additionally demonstrate the use of initiator ...
In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic
Plant growth throughout the world is often limited by unfavourable environmental conditions. This paper reports results of a study on long- and short-term osmotic stress (−0.5 MPa) followed by a recovery on in vitro translational capacity of polysomes and on the composition of polysome-associated proteins in germinating pea (Pisum sativum L.) seeds. Here we show that, under osmotic stress, cytoskeleton-bound polysomes were charaterized by the highest translation activity, which may be indicative of an important role that this population of polysomes plays in the synthesis of the so-called stress proteins. We also find out that in response to osmotic stress, new proteins (22.01, 96.47 and 105.3 kDa), absent in the unstressed sample, associated with the total pool of polysomes, whereas the protein of 22.95 kDa, which was present in the embryonic tissue of seeds germinating under unstressed conditions, disappeared. These changes may have affected both the stability and the translational ...
TY - JOUR. T1 - A fission yeast general translation factor reveals links between protein synthesis and cell cycle controls. AU - Grallert, B. AU - Kearsey, SE. AU - Lenhard, M. AU - Carlson, CR. AU - Nurse, P. AU - Boye, E. AU - Labib, K. PY - 2000. Y1 - 2000. N2 - In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects ill B-cyclin function or expression. These mutations define a single gene which we call ded1, We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 land to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited ...
MOTIVATION: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. RESULTS: We present a statistical framework and an analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy ...
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One of the hallmark metabolic responses to stress (i.e. injury, sepsis, surgery) is catabolism. Generally speaking, the degree of catabolism is proportional to the degree of stress. Catabolism is caused by increased breakdown of skeletal muscle. The subsequent release of amino acids provides some of the substrate for increased hepatic gluconeogenesis.. During periods of stress, the overall nitrogen balance is negative. However, there is conflicting data regarding the degree of skeletal muscle protein synthetic activity. Studies have reported reduced skeletal muscle protein synthesis after open cholecystectomy, decreased hepatic protein synthesis that becomes directly proportional to the duration of surgery, and a reduction in the protein synthesis within tissues that have rapidly replicating cells. Other studies postulate that the negative nitrogen balance is the result of protein breakdown that exceeds the increased protein synthetic rate.. Stress hormones like cortisol and cytokines such as ...
When faced with the selection pressure of chronic mTORC1 and mTORC2 inhibition by AZD8055, SW620 cells remodelled mTOR signalling to allow them to continue to proliferate. We anticipated that this adaptation might involve a switch from cap-dependent to IRES-dependent translation because: (1) compensatory IRES-dependent translation is seen upon inhibition of cap-dependent translation (supplementary material Fig. S2) (Svitkin et al., 2005); (2) this was observed upon acute AZD8055 treatment (Fig. 1D); and (3) some oncogenes are translated by IRES-dependent mechanisms (Stoneley and Willis, 2004). However, IRES-dependent translation was not upregulated in SW620:8055R cells. Rather the cells adapted to chronic mTORC1 and mTORC2 inhibition by amplifying eIF4E (Fig. 4) to increase eIF4E protein levels, thereby maintaining or even increasing cap-dependent translation (Fig. 5). RNAi to eIF4E revealed that SW620:8055R cells remained addicted to the increased expression of eIF4E to maintain AZD8055 ...
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Cyclopentenone prostaglandins are potent inhibitors of virus replication. The antiviral activity has been associated with the induction of 70-kDa heat shock protein (HSP70) synthesis. In this report, we describe that in African green monkey kidney cells infected with Sendai virus (SV) and treated with prostaglandin A1 (PGA1), SV protein synthesis was selectively blocked as long as HSP70 was being synthesized by the host cell. The block appeared to be at the translational level, as indicated by the following (i) PGA1 had no effect on SV primary transcription, and a dramatic decrease in the abundance of SV mRNA occurred only at later stages of infection; and (ii) treatment with PGA1 started at 6 h postinfection, at which time SV mRNA had already accumulated in infected cells, did not suppress the levels of NP mRNA, but it reduced the amount of ribosome-bound NP mRNA and caused a dramatic decrease in the level of genomic RNA. The PGA1-induced block of SV protein synthesis appeared to be cell ...
BACKGROUND: Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. RESULTS: The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from
Principal Investigator:SUYAMA Akira, Project Period (FY):2011-04-01 - 2016-03-31, Research Category:Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Project Area:Synthetic biology for the comprehension of biomolecular networks
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TY - CHAP. T1 - Cell-free Synthesis of Macromolecular Complexes. AU - Botte, Mathieu. AU - Deniaud, Aurelien. AU - Schaffitzel, Christiane. PY - 2016/5/11. Y1 - 2016/5/11. N2 - Cell-free protein synthesis based on E. coli cell extracts has been described for the first time more than 50 years ago. To date, cell-free synthesis is widely used for the preparation of toxic proteins, for studies of the translation process and its regulation as well as for the incorporation of artificial or labeled amino acids into a polypeptide chain. Many efforts have been directed towards establishing cell-free expression as a standard method for gene expression, with limited success. In this chapter we will describe the state-of-the-art of cell-free expression, extract preparation methods and recent examples for successful applications of cell-free synthesis of macromolecular complexes.. AB - Cell-free protein synthesis based on E. coli cell extracts has been described for the first time more than 50 years ago. To ...
GPR41 is a G protein-coupled receptor activated by short chain fatty acids. The gene encoding GPR41 is located immediately downstream of a related gene encoding GPR40, a receptor for long chain fatty acids. Expression of GPR41 has been reported in a small number of cell types, including gut enteroendocrine cells and sympathetic ganglia, where it may play a role in the maintenance of metabolic homeostasis. We now demonstrate that GPR41, like GPR40, is expressed in pancreatic beta cells. Surprisingly, we found no evidence for transcriptional control elements or transcriptional initiation in the intergenic GPR40-GPR41 region. Rather, using 5-rapid amplification of cDNA ends analysis, we demonstrated that GPR41 is transcribed from the promoter of the GPR40 gene. We confirmed this finding by generating bicistronic luciferase reporter plasmids, and we were able to map a potential internal ribosome entry site-containing region to a 2474-nucleotide region of the intergenic sequence. Consistent with this, we
What is the most efficient translation initiation signal? Does the small subunit ribosome do the scanning for the start codon or can a fully formed ribosome scan for start codon, too? When to scan from the 5 end and when to scan from the middle of 5UTR (internal ribosomal entry)? If secondary structure embedding start codon (or Shine-Dalgarno sequence or Kozak consensus) can obscure the translation initiation signal, how would highly expressed genes avoid this? What is the best way of measuring gene expression experimentally or bioinformatically? Do translation initiation efficiency affect elongation efficiency/accuracy? How to measure translation elongation efficiency/accuracy? Will a few closely spaced minor codons severely affect translation elongation? What is the optimal translation stop signal? How do release factors decode the stop signal? How the relative concentration and decoding capacity of different release factors affect stop codon usage? How frequent do tRNAs misread stop codons ...
TY - JOUR. T1 - Human lymphocyte messenger RNA activity profiles in type I and type II diabetes. T2 - A tool for classification of metabolic disease. AU - Mariash, C. N.. AU - Burmeister, L. A.. PY - 1988/12/1. Y1 - 1988/12/1. N2 - We have previously used rat hepatic messenger ribonucleic acid (mRNA) activity profiles to categorize various pathophysiologic states. To test the hypothesis that similar techniques can be used to categorize disease states in humans, we examined the mRNA activity profiles by using in vitro translational assays of Ficoll-Hypaque-separated mononuclear cells obtained from six normal volunteers, six patients with type I diabetes, and five patients with type II diabetes as examples of different disease states. Translated proteins were labeled with sulfur 35-labeled methionine, separated by two-dimensional gel electrophoresis, and quantitated by videodensitometry of autoradiographs derived from the two-dimensional gels. Of approximately 160 quantitated mRNAs, the levels of ...
Surveying the relative impact of mRNA features on local ribosome profiling read density in 28 datasets. Patrick OConnor , Dmitry Andreev , Pavel Baranov doi: http://dx.doi.org/10.1101/018762 Ribosome profiling is a promising technology for exploring gene expression. However, ribosome profiling data are characterized by a substantial number of outliers due to technical and biological factors. Here…
Enucleated mouse 1-cell embryos arrest development at the 2-cell stage following transplantation of cleavage stage nuclei. Earlier studies employing one-dimensional protein gel electrophoresis failed to reveal obvious differences in gene expression in the manipulated embryos that might account for this block. We report here the results of a quantitative, two-dimensional gel electrophoretic analysis that reveals at least 50 alterations in protein synthesis in the 8--|1-cell nuclear transplant embryos. Approximately half of these alterations involve proteins that normally decrease in synthesis between the 2-cell and 8-cell stages and half involve proteins that are synthesized constitutively between these two stages. These results are the first to reveal significant biochemical alterations that accompany the morphological and cytological differences previously described and indicate that the 8-cell stage nucleus is unable to completely recapitulate the normal progression of changes in protein
Oxygen and glucose metabolism play pivotal roles in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death. Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and
Glycogen synthase kinase 3 (GSK3) has long been known as a signaling component in insulin regulation of metabolism and, more recently, as a key part of the Wnt signaling pathway regulating cell proliferation, cell fate, and other processes during development. Unlike most other kinases, GSK3 is constitutively active and is regulated by inhibition. Full expression of this constitutive activity in the mammalian enzyme appears to require phosphorylation of a tyrosine residue in the activation loop of GSK3, which the enzyme accomplishes by intramolecular autophosphorylation, even though the kinase phosphorylates strictly serine and threonine residues on its exogenous substrates. Lochhead et al. explored the mechanism by which this switch in residue specificity is possible. Tagged GSK-3β synthesized in a rabbit reticulocyte lysate translation system showed rapid autophosphorylation that was inhibited by inhibitors of the molecular chaperone protein Hsp90. This chaperone-assisted tyrosine kinase ...
Abstract: Among various membrane-bound polyribosomes from chicken embryos the polyribosomes loosely bound with membranes proved to be highly active in synthesis of total proteins as well as of collagens in vitro. These data suggest that polyribosomes loosely bound with membranes were not an impurity of free polyribosomes in the total preparation of the membrane-bound polyribosomes. These polyribosomes constituted a definite class of polyribosomes active in the synthesis of secreted proteins (i.e. of collagen). In polyribosome fractions identified by their size (monosomes, light and heavy polyribosomes) all the three fractions of loosely-bound polyribosomes as well as light and heavy fractions of tightly-bound polyribosomes were active in synthesis of total proteins. Differences between tightly-and loosely-bound polyribosomes were noted also in studies of cell-free synthesis of collagen proteins. Heavy fractions of tightly-bound polyribosomes were the most active in synthesis of these proteins, ...
Microsome; Translation machinery The ribosome is the central component of the protein synthesis machinery in the cell. It contains both RNAand protein and is composed of two subunits. Ribosomes...
Most of our current knowledge about gene regulation is based on studies of mRNA levels, despite both the greater functional importance of protein abundance, and evidence that post-transcriptional regulation is pervasive. However, understanding the molecular basis of regulatory variation within and between species may prove very useful. Indeed, the majority of identified human disease-risk alleles lie in non-coding regions of the genome, suggesting that they affect gene regulation. Until recently, the lack of performant high-throughput methods for detecting protein abundance hampered the in-depth study of gene regulation. However, a new method known as ribosome profiling has enabled us to study divergence in the regulation of translation.. Ribosome profiling or riboprofiling involves the construction of two RNA-seq libraries: one measuring mRNA abundance (the mRNA fraction), and the second capturing the portion of the transcriptome that is actively being translated by ribosomes (the Ribo ...
Over the past few years, there has been a growing interest in the interconnection between translation and metabolism. Important oncogenic pathways, like those elicited by c-Myc transcription factor and mTOR kinase, couple the activation of the translational machinery with glycolysis and fatty acid synthesis. Eukaryotic initiation factor 6 (eIF6) is a factor necessary for 60S ribosome maturation. eIF6 acts also as a cytoplasmic translation initiation factor, downstream of growth factor stimulation. eIF6 is up-regulated in several tumor types. Data on mice models have demonstrated that eIF6 cytoplasmic activity is rate-limiting for Myc-induced lymphomagenesis. In spite of this, eIF6 is neither transcriptionally regulated by Myc, nor post-transcriptionally regulated by mTOR. eIF6 stimulates a glycolytic and fatty acid synthesis program necessary for tumor growth. eIF6 increases the translation of transcription factors necessary for lipogenesis, such as CEBP/β, ATF4 and CEBP/δ. Insulin stimulation leads
An miRNA-Mediated Therapy for SCA6 Blocks IRES-Driven Translation of the CACNA1A Second Cistron Researchers identified miR-3191-5p as a microRNA (miRNA) that targeted CACNA1A internal ribosomal entry site (IRES) and preferentially inhibited the CACNA1A IRES-driven translation of α1ACT in an Argonaute 4-dependent manner. They found that eukaryotic initiation factors (eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhance α1ACT translation. [Sci Transl Med] Abstract Neurons Differentiated from Transplanted Stem Cells Respond Functionally to Acoustic Stimuli in the Awake Monkey Brain Researchers examined whether neurons differentiated from transplanted stem cells can integrate into the host neural network and function in awake animals, a goal of transplanted stem cell therapy in the brain. [Cell Rep] Full Article , Graphical Abstract Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy One million C2C12 ...
Kits and reagents for cell-free protein expression in just a few hours using mRNA templates in translational systems, or DNA template (plasmid DNA or PCR fragments) in coupled transcription and translation systems.
In response to viral pathogens, the host upregulates antiviral genes that suppress translation of viral mRNAs. However, induction of such antiviral responses may not be exclusive to viruses, as the pathways lie at the intersection of broad inflammatory networks that can also be induced by bacterial pathogens. Using a model of Gram-negative sepsis, we show that propagation of kidney damage initiated by a bacterial origin ultimately involves antiviral responses that result in host translation shutdown. We determined that activation of the eukaryotic translation initiation factor 2-α kinase 2/eukaryotic translation initiation factor 2α (Eif2ak2/Eif2α) axis is the key mediator of translation initiation block in late-phase sepsis. Reversal of this axis mitigated kidney injury. Furthermore, temporal profiling of the kidney translatome revealed that multiple genes involved in formation of the initiation complex were translationally altered during bacterial sepsis. Collectively, our findings imply ...
Mitochondria have their own transcriptional and translational apparatus, even though they produce only a handful of proteins, therefore most of the proteins are imported from the cytoplasm. Trancription, translation and protein insertion into the membrane are interconnected: translational activators regulating mitochondrial translation are interacting with mitochondrial RNA polymerase via Nam1p and Sls1p proteins (Bryan et al. Genetics 2002), Puf proteins connect cytoplasmic translation and protein import into mitochondria by direct interaction with Tom20 subunit of the TOM protein import channel (Saint-Georges et al. PLoS ONE 2008 ...
The study is published in the Aug. 14 online edition of the journal Nature Chemical Biology.. This work began when University of Toronto scientists exploring the origins of Salmonellas virulence identified three genes that were clear players in the process. These three genes - called YjeK, PoxA and EF-P - were unusual in this context. Genes that confer virulence in bacteria typically have a specific job, such as producing toxins or transporters. But these three virulence genes all looked like they should have a role in the protein synthesis machinery - which is Ibbas expertise.. Under normal circumstances in cells, an enzyme will select amino acids in the cell and place them on a molecule called transfer RNA, or tRNA, which leads to translation of the genetic code into proteins.. In Salmonella cells, these steps are similar, but with a few surprising twists, Ibba said. He and colleagues confirmed that the YjeK gene makes beta lysine, and showed that the PoxA gene takes that beta lysine and ...
The demand for recombinant therapeutic proteins grows larger every year. These medicines are used for treatment of myriad disorders and conditions, including cancer, lysosomal storage diseases, and diabetes. The vast majority of therapeutic proteins are glycoproteins, meaning that they have short, distinctive carbohydrate chains attached to particular amino acids of the protein backbone. These carbohydrate chains are often required for proper function or targetting of the protein.. Many different eukaryotic hosts, including yeast, hamster and insect cell cultures, plants, chickens, and even lactating mammals have been engineered to produce therapeutic proteins at industrial scale. The issue is that while the core protein synthesis machinery is more-or-less conserved across eukaryotic kingdoms, the carbohydrate chains that are added to the proteins by the cell are highly variable. Even the glycosylation patterns of mammalian cell cultures can prove difficult to control. If the protein is produced ...
The results of the genome-wide ribosome profiling analysis significantly redefine our understanding of the mode of action of macrolide antibiotics. In contrast to the previously prevailing view, and in agreement with the more recent proteomic and biochemical experiments, ribosome profiling clearly reveals these antibiotics as context- and thus protein-specific inhibitors of translation. Synthesis of the protein is interrupted not simply when the nascent chain reaches the site of the drug binding in the NPET but also when the drug-bound ribosome comes across a sequence of codons specifying specific combinations of amino acids.. A striking finding that emerged from the results of the profiling analysis and biochemical experiments is that the sites of macrolide-dependent translation arrest are defined primarily not by the sequence of the nascent chain juxtaposed with the antibiotic in the NPET but by the nature of the amino acid residues in the PTC: specifically, the C-terminal amino acids of the ...
TY - JOUR. T1 - RNA trafficking and local protein synthesis in dendrites. T2 - an overview.. AU - Martin, Kelsey C.. AU - Zukin, R. Suzanne. PY - 2006/7/5. Y1 - 2006/7/5. N2 - It is now widely accepted that mRNAs localize to dendrites and that translation of these mRNAs is regulated in response to neuronal activity. Recent studies have begun to reveal the underpinnings of these processes and to underscore the importance of local protein synthesis to synaptic remodeling and plasticity. When Steward and Levy (1982) first reported their observation of polyribosomes at the base of spines, the prevailing view was that all proteins were synthesized in the cell body and then transported to distal compartments of neurons. Steward and Levys discovery, however, raised the intriguing possibility that mRNAs could be transported to synapses and locally translated in response to synaptic stimulation. This provided an elegant mechanism for spatially restricting gene expression within the neuron, such that ...
Homologous recombination. The mouse Eif4ebp2 gene was obtained by screening a λ FixII 129/SvJ mouse genomic library (Tsukiyama-Kohara et al., 2001). The targeting vector consisted of a 4.9 kb XbaI Eif4ebp2 genomic fragment upstream of exon 2, a pTK-Neo cassette (pMC1neo; Stratagene, La Jolla, CA), and a 1.2 kb AflIII-BclI Eif4ebp2 genomic fragment downstream of exon 2. Electroporation of the linearized vector (NotI) into 129/Sv embryonic stem (ES) cell line J1 (Li et al., 1992) and selection of G418-resistant transformants were performed as described previously (You-Ten et al., 1997). G418-resistant colonies were analyzed for homologous recombination by Southern blot analysis.. Mutant mice. Generation of chimeric and mutant mice was performed as described previously (You-Ten et al., 1997). Genotyping was performed by Southern blot analysis with probes derived from a 1.8 kb XbaI fragment located upstream of the targeting vector, a fragment derived from the 3′ untranslated region of Eif4ebp2, ...
When programmed with yeast prepro-α-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-α-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp-α-F3, 27.5 kDa). Glycosylation of the membrane specific pp-α-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-α-F0), whereas the primary translation product pp-α-F cyt is not affected. Likewise, only the glycosylated pp-α-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between PP-α-F0 and pp-α-F cyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due ...
Efficient neuronal function depends on the continued modulation of the local neuronal proteome. Local protein synthesis plays a central role in tuning the neuronal proteome at specific neuronal regions. Various aspects of translation such as the localization of translational machinery, spatial spread of the newly translated proteins, and their site of action are carried out in specialized neuronal subcompartments to result in a localized functional outcome. In this review, we focus on the various aspects of these local translation compartments such as size, biochemical and organelle composition, structural boundaries, and temporal dynamics. We also discuss the apparent absence of definitive components of translation in these local compartments and the emerging state‐of‐the‐art tools that could help dissecting these conundrums in greater detail in the future. ...
Cambio, distributor of molecular biology reagents and consumables to scientific research laboratories within the UK, has added an innovative new product to its portfolio. ARTseq™ (Active mRNA Translation) ribosome profiling kits, produced by Epicentre, enable users to create RNA sequence libraries from ribosome-protected mRNA. This novel technique is used to investigate translational control, measure gene expression, identify translation start sites and predict protein abundance, making them ide
Mitochondria do not have IF1 (all mitochondria), and unlike mammals, yeast ones do not have IF3! Also they seem to use mitochondria-specific initiation factor AEP3. Do mammals have AEP3 homologue? Worth checking... To make things more complicated, mitochondria have their own special mRNA-specific factors... and again, there is a catch. Yeast mRNA have long 5 UTRs (untranslated regions), and mammals have short, they basically have leaderless mRNAs (Id love to see a good reference for that! THIS is a little bit out of date...) - therefore I would expect that these mRNA-specific IFs work differently in yeast in mammals. So yeast and mammalian mitochondrial translation seems to have very, very different translational apparatus. Isnt is weird ...
Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and ...
BACKGROUND: The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNDelta1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein. METHODS: Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCNDelta1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. RESULTS: Both are translated, but higher levels of protein were seen with MYCNDelta1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNDelta1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained
During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local ...
Knowledge of the function of the mammalian eIF2α phosphatases stems from their homology to a viral protein. In 1994, Joany Chou and Bernard Roizman discovered the function of the γ134.5 gene of herpes simplex virus: mutants lacking a functional γ134.5 gene failed to escape the shutdown of protein synthesis that followed viral infection [45]. They noted that the carboxy-terminal domain of γ134.5 is homologous to the carboxy-terminal domain of the product of the growth arrest and DNA damage gene Gadd34 (now PPP1R15A) [45] and indeed later showed that the carboxy-terminal domain of Gadd34 can substitute for the homologous domain of γ134.5 [46]. Chou and colleagues found that shutdown of protein synthesis following viral infection was mediated by PKR which phosphorylated eIF2α [47]. The Roizman laboratory then performed an unbiased yeast two-hybrid screen and found that Gadd34 and γ134.5 interacted with PP1c [48]. The functional relevance of this interaction was supported by the finding that ...
The programme is divided into units of study called modules which are assigned credits. The credit rating of a module is proportional to the total workload, with 1 credit being nominally equivalent to 10 hours of work.. Students on the MA Translation Studies take a selection of modules amounting to 180 credits in total. This includes a compulsory Translation Dissertation (60 credits) and two compulsory modules: Translation Theory (30 credits) and The Practice of Translation (30 credits). The remaining 60 credits are chosen from our optional modules, see below.. The modules we outline here provide examples of what you can expect to learn on this degree course based on recent academic teaching. The precise modules available to you in future years may vary depending on staff availability and research interests, new topics of study, timetabling and student demand.. ...
The fidelity of translation depends on accurate selection of the correct reading frame during initiation. In eukaryotes, this process involves at least 11 eukaryotic initiation factors (eIFs). Met‐tRNAiMet forms a ternary complex with eIF2 and GTP, which together with eIF1, eIF1A and eIF3 binds to the 40S ribosomal subunit to form a 43S preinitiation complex. After loading onto the mRNAs 5′ end in a process requiring eIFs 4A, 4B and 4F, the 43S complex scans downstream until it encounters an AUG triplet in a favorable context GCC(A/G)CCAUGG (in which the nucleotides at the −3 and +4 positions are the most important; Kozak, 1991), stops and forms a stable 48S complex with established codon-anticodon base pairing in the P site. These two context nucleotides are important features of mammalian mRNAs but differ in sequence and importance in other eukaryotes. Subsequent joining of a 60S subunit is mediated by eIF5 (which induces hydrolysis of eIF2‐bound GTP and dissociation of eIF2‐GDP ...
The IRESite database presents information about the experimentally studied IRES (Internal Ribosome Entry Site) segments. IRES regions are known to attract eukaryotic ribosomal translation initiation complex and thus promote translation initiation independently of the presence of the commonly utilized 5-terminal 7mG cap structure. It is not yet clear whether the activity could be attributed to a common sequence or to a common secondary structure present in them. Such IRES regions were found in a broad range of +RNA viruses and in some eukaryotic cellular mRNAs. Certain IRESs have been predicted based on the fact that closely related viruses should share its features and indeed, their sequences are in some regions conserved enough to allow identification of a new IRES. In contrast, cellular IRESs do not have almost anything in common in terms of their sequence or secondary structure. Therefore, cellular IRESs cannot be predicted. Either way, IRESite is focused only on IRESs which have been at ...
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