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Chemistry & Biology Jun 2005The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability...
The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability to selectively interfere with it. To inhibit protein synthesis with spatial and temporal control, we have developed a photo-releasable anisomycin compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), that can be removed through exposure to UV light. The area of protein synthesis inhibition can be restricted to a small light-exposed region or, potentially, the volume of two-photon excitation if a pulsed IR laser is the light source. We have tested the compound's effectiveness with an in vitro protein-translation system, CHO cells, HEK293 cells, and neurons. The photo-released anisomycin can inhibit protein synthesis in a spatially restricted manner, which will enable the specific inhibition of protein synthesis in subsets of cells with temporal and spatial precision.
Topics: Animals; Anisomycin; Cell Line; Cricetinae; Genes, Reporter; Humans; Light; Neurons; Photolysis; Protein Biosynthesis; Rats; Spectrum Analysis; Ultraviolet Rays
PubMed: 15975514
DOI: 10.1016/j.chembiol.2005.04.018 -
Cold Spring Harbor Perspectives in... Nov 2018One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single... (Review)
Review
One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation.
Topics: Epitopes; Gene Expression Regulation; Humans; Molecular Imaging; Peptide Chain Elongation, Translational; Protein Biosynthesis; RNA, Messenger
PubMed: 30385605
DOI: 10.1101/cshperspect.a032078 -
Trends in Cell Biology Dec 2015Cell size is an important physiological trait that sets the scale of all biosynthetic processes. Although physiological studies have revealed that cells actively... (Review)
Review
Cell size is an important physiological trait that sets the scale of all biosynthetic processes. Although physiological studies have revealed that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained unclear. Here we review recent progress in identifying the molecular mechanisms of cell size control. We focus on budding yeast, where cell growth dilutes a cell cycle inhibitor to couple growth and division. We discuss a new model for size control based on the titration of activator and inhibitor molecules whose synthesis rates are differentially dependent on cell size.
Topics: Animals; Cell Cycle; Cell Differentiation; Cell Proliferation; Cell Size; Humans; Protein Biosynthesis
PubMed: 26573465
DOI: 10.1016/j.tcb.2015.10.006 -
ELife Dec 2020Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional...
Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited because they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme-substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.
Topics: Biophysical Phenomena; DNA Replication; Hydrolysis; Kinetics; Models, Biological; Protein Biosynthesis; Substrate Specificity
PubMed: 33357378
DOI: 10.7554/eLife.60415 -
Nucleic Acids Research May 2021Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits...
Ribosomes are evolutionary conserved ribonucleoprotein complexes that function as two separate subunits in all kingdoms. During translation initiation, the two subunits assemble to form the mature ribosome, which is responsible for translating the messenger RNA. When the ribosome reaches a stop codon, release factors promote translation termination and peptide release, and recycling factors then dissociate the two subunits, ready for use in a new round of translation. A tethered ribosome, called Ribo-T, in which the two subunits are covalently linked to form a single entity, was recently described in Escherichia coli. A hybrid ribosomal RNA (rRNA) consisting of both the small and large subunit rRNA sequences was engineered. The ribosome with inseparable subunits generated in this way was shown to be functional and to sustain cell growth. Here, we investigated the translational properties of Ribo-T. We analyzed its behavior during amino acid misincorporation, -1 or +1 frameshifting, stop codon readthrough, and internal translation initiation. Our data indicate that covalent attachment of the two subunits modifies the properties of the ribosome, altering its ability to initiate and terminate translation correctly.
Topics: Codon, Terminator; Frameshifting, Ribosomal; Peptide Chain Initiation, Translational; Peptide Chain Termination, Translational; Protein Biosynthesis; RNA, Transfer; Ribosomes
PubMed: 33950196
DOI: 10.1093/nar/gkab259 -
Angewandte Chemie (International Ed. in... Feb 2020By transplanting identity elements into E. coli tRNA , we have engineered an orthogonal initiator tRNA (itRNA ) that is a substrate for Methanocaldococcus jannaschii...
By transplanting identity elements into E. coli tRNA , we have engineered an orthogonal initiator tRNA (itRNA ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.
Topics: Amino Acids; Humans; Protein Biosynthesis; Protein Engineering
PubMed: 31828898
DOI: 10.1002/anie.201914671 -
Methods in Molecular Biology (Clifton,... 2018We present an alternative production platform for the synthesis of complex proteins. Apart from conventionally applied protein production using engineered mammalian cell...
We present an alternative production platform for the synthesis of complex proteins. Apart from conventionally applied protein production using engineered mammalian cell lines, this protocol describes the preparation and principle of cell-free protein synthesis systems based on CHO cell lysates. The CHO cell-free system contains endogenous microsomes derived from the endoplasmic reticulum, which enables a direct integration of membrane proteins into a nature like milieu and the introduction of posttranslational modifications. Different steps of system development are described including the cultivation of CHO cells, cell harvesting and cell disruption to prepare translationally active CHO cell lysates. The requirements for DNA templates and the generation of linear DNA templates suitable for the CHO cell-free reaction is further depicted to underline the opportunity to produce different protein variants in a short period. This experimental setup provides a basis for high-throughput applications. The productivity of the CHO cell-free systems is further increased by using a non-canonical translation initiation due to the attachment of an internal ribosomal entry site of the Cricket paralysis virus (CRPV IRES) to the 5´ UTR of the desired gene. In this way, a direct interaction of the IRES structure with the ribosome facilitates a translation factor independent initiation of translation. Cell-free reactions were performed in fast and efficient batch reactions leading to protein yields up to 40 μg/mL. The reaction format was further adjusted to a continuous exchange CHO cell-free reaction (CHO CECF) to prolong reaction time and thereby increase the productivity of the cell-free systems. Finally, protein yields up to 1 g/L were obtained. The CHO CECF system represents a sophisticated resource to address structural and functional aspects of difficult-to-express proteins in fundamental and applied research.
Topics: Animals; Biotechnology; CHO Cells; Cell-Free System; Cricetinae; Cricetulus; Internal Ribosome Entry Sites; Membrane Proteins; Protein Biosynthesis; Protein Processing, Post-Translational
PubMed: 30242694
DOI: 10.1007/978-1-4939-8730-6_19 -
Current Opinion in Clinical Nutrition... May 2008To highlight recent studies that have examined the cell-signalling mechanisms responsible for the amino acid (primarily leucine and the essential amino acids)... (Review)
Review
PURPOSE OF REVIEW
To highlight recent studies that have examined the cell-signalling mechanisms responsible for the amino acid (primarily leucine and the essential amino acids) stimulation of human skeletal muscle protein synthesis.
RECENT FINDINGS
Ingestion of a leucine-enriched essential amino acid nutrient solution rapidly and potently activates the mammalian target of rapamycin signalling pathway and protein synthesis in human skeletal muscle. Further, mTOR signalling and muscle protein synthesis are enhanced when leucine-enriched nutrients are ingested following resistance exercise. The addition of leucine to regular meals may improve the ability of feeding to stimulate protein synthesis in old human muscle.
SUMMARY
Leucine and essential amino acids appear to stimulate human muscle protein synthesis primarily by activating the mammalian target of rapamycin signalling pathway. How human muscle cells sense an increase in leucine and/or essential amino acids to activate mammalian target of rapamycin signalling is currently unknown. Recent work, however, suggests that the kinases hVps34 and MAP43K may be involved. Leucine-enriched essential amino acid ingestion, in combination with resistance exercise in some cases, may be a useful intervention to promote mTOR signalling and protein synthesis in an effort to counteract a variety of muscle wasting conditions (e.g. sarcopenia, cachexia, AIDS, inactivity/bed rest, sepsis, kidney failure, and trauma).
Topics: Humans; Leucine; Muscle Proteins; Nutritional Physiological Phenomena; Peptide Chain Elongation, Translational; Protein Biosynthesis; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Weight Lifting
PubMed: 18403916
DOI: 10.1097/MCO.0b013e3282fa17fb -
Trends in Plant Science Apr 2017MicroRNAs (miRNAs) function to post-transcriptionally regulate target RNAs, including long non-coding RNAs and mRNAs. A recent study demonstrates that Arabidopsis miRNAs... (Review)
Review
MicroRNAs (miRNAs) function to post-transcriptionally regulate target RNAs, including long non-coding RNAs and mRNAs. A recent study demonstrates that Arabidopsis miRNAs are enriched at the rough endoplasmic reticulum (ER). This enrichment is a surprise, given that most known miRNA targets are not expected to be translated at the rough ER.
Topics: Arabidopsis; Endoplasmic Reticulum; MicroRNAs; Protein Biosynthesis; RNA, Messenger
PubMed: 28320581
DOI: 10.1016/j.tplants.2017.03.002 -
Proteomics Mar 2014Acute biological effects caused by the exposure to high doses of radiation, either ionizing or nonionizing, are relatively well-known but the delayed effects, occurring... (Review)
Review
Acute biological effects caused by the exposure to high doses of radiation, either ionizing or nonionizing, are relatively well-known but the delayed effects, occurring decades after exposure, are difficult to predict. The knowledge of the acute and delayed effects of the low doses of ionizing radiation (e.g. bystander effect) or nonionizing radiation (e.g. radiation emitted by wireless communication devices) is not yet reliably established. Often the acute effects of low doses are small and difficult to discover and replicate in scientific studies. Chronic effects of prolonged exposures to low-dose radiation for decades are virtually unknown and often not possible to predict on the basis of the knowledge gained from acute exposures to high doses of radiation. Physiological significance of the biological effects induced by low doses of radiation is not known. The same lack of predictability of outcomes applies to the delayed effects of high-dose radiation exposures. Proteomics, supplemented with other "omics" techniques, might be the best way forward to find out the target molecules of radiation, the biomarkers of radiation exposure and the physiological and health significance of the acute and delayed biological effects caused by the exposures to high- and low-dose radiation. However, the currently available database of radiation effects on proteomes is far too small to be useful in formulation of new hypotheses concerning health consequences of radiation exposures.
Topics: Dose-Response Relationship, Radiation; Humans; Protein Biosynthesis; Proteomics; Radiation; Signal Transduction
PubMed: 24376023
DOI: 10.1002/pmic.201300390