Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function

AbstractDecoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

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Exploiting RNA thermometer-driven molecular bioprocess control as a concept for heterologous rhamnolipid production

Scientific Reports ◽

10.1038/s41598-021-94400-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Philipp Noll ◽

Chantal Treinen ◽

Sven Müller ◽

Lars Lilge ◽

Rudolf Hausmann ◽

...

Keyword(s):

High Titer ◽

Production Capacity ◽

Batch Process ◽

Product Formation ◽

Effect Of Temperature ◽

Industrial Biotechnology ◽

Temperature Sensitive ◽

Translation Control ◽

Rhamnolipid Production ◽

Rna Thermometer

AbstractA key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.

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Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)77687-x ◽

1988 ◽

Vol 263 (36) ◽

pp. 19662-19669

Author(s):

T L Solomon ◽

L R Solomon ◽

L S Gay ◽

P A Rubenstein

Keyword(s):

Aspartic Acid ◽

Site Specific

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Phosphorylation of the Regulators, a Complex Facet of NF-κB Signaling in Cancer

Biomolecules ◽

10.3390/biom11010015 ◽

2020 ◽

Vol 11 (1) ◽

pp. 15

Author(s):

Aishat Motolani ◽

Matthew Martin ◽

Mengyao Sun ◽

Tao Lu

Keyword(s):

Transcription Factor ◽

Cardiovascular Diseases ◽

Cancer Progression ◽

Nuclear Factor Kappa B ◽

Nuclear Factor ◽

Malignant Diseases ◽

Future Perspectives ◽

Post Translational Modifications ◽

Site Specific

The nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor central to inflammation and various malignant diseases in humans. The regulation of NF-κB can be influenced by a myriad of post-translational modifications (PTMs), including phosphorylation, one of the most popular PTM formats in NF-κB signaling. The regulation by phosphorylation modification is not limited to NF-κB subunits, but it also encompasses the diverse regulators of NF-κB signaling. The differential site-specific phosphorylation of NF-κB itself or some NF-κB regulators can result in dysregulated NF-κB signaling, often culminating in events that induce cancer progression and other hyper NF-κB related diseases, such as inflammation, cardiovascular diseases, diabetes, as well as neurodegenerative diseases, etc. In this review, we discuss the regulatory role of phosphorylation in NF-κB signaling and the mechanisms through which they aid cancer progression. Additionally, we highlight some of the known and novel NF-κB regulators that are frequently subjected to phosphorylation. Finally, we provide some future perspectives in terms of drug development to target kinases that regulate NF-κB signaling for cancer therapeutic purposes.

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Molecular modulation of calcium oxalate crystallization

AJP Renal Physiology ◽

10.1152/ajprenal.00136.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. F1123-F1132 ◽

Cited By ~ 57

Author(s):

James J. De Yoreo ◽

S. Roger Qiu ◽

John R. Hoyer

Keyword(s):

Molecular Modeling ◽

Calcium Oxalate ◽

Stone Formation ◽

Energy Levels ◽

Critical Role ◽

Specific Interactions ◽

Crystal Surfaces ◽

Site Specific

Calcium oxalate monohydrate (COM) is the primary constituent of the majority of renal stones. Osteopontin (OPN), an aspartic acid-rich urinary protein, and citrate, a much smaller molecule, are potent inhibitors of COM crystallization at levels present in normal urine. Current concepts of the role of site-specific interactions in crystallization derived from studies of biomineralization are reviewed to provide a context for understanding modulation of COM growth at a molecular level. Results from in situ atomic force microscopy (AFM) analyses of the effects of citrate and OPN on growth verified the critical role of site-specific interactions between these growth modulators and individual steps on COM crystal surfaces. Molecular modeling investigations of interactions of citrate with steps and faces on COM crystal surfaces provided links between the stereochemistry of interaction and the binding energy levels that underlie mechanisms of growth modification and changes in overall crystal morphology. The combination of in situ AFM and molecular modeling provides new knowledge that will aid rationale design of therapeutic agents for inhibition of stone formation.

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Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

Journal of Bacteriology ◽

10.1128/jb.01613-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1565-1572 ◽

Cited By ~ 82

Author(s):

Venkata Ramana Vepachedu ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Small Molecules ◽

Spore Germination ◽

Dipicolinic Acid ◽

Temperature Sensitive ◽

Wild Type ◽

Ph Optimum ◽

Inner Membrane ◽

Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

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V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1363 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1363-1370 ◽

Cited By ~ 31

Author(s):

K M Sheehan ◽

M R Lieber

Keyword(s):

Genome Stability ◽

Lymphoid Cells ◽

Chromosomal Translocations ◽

Synaptic Inhibition ◽

Site Specific ◽

Coding Sequences ◽

Specific Process ◽

A Site ◽

Joint Resolution

V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.

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Metabolic adaptation of renal carbohydrate metabolism. IV. The use of site-specific liver gluconeogenesis inhibitors to ascertain the role of renal gluconeogenesis (1)

Archives Internationales de Physiologie de Biochimie et de Biophysique ◽

10.3109/13813459109146928 ◽

1991 ◽

Vol 99 (3) ◽

pp. 237-242 ◽

Cited By ~ 1

Author(s):

L. Garcia-salguero ◽

F. Aranda ◽

J. Peragon ◽

F. J. Corpas ◽

J. A. Lupiañez

Keyword(s):

Carbohydrate Metabolism ◽

Metabolic Adaptation ◽

Site Specific ◽

Renal Gluconeogenesis

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A High Copy Suppressor Screen Reveals Genetic Interactions Between BET3 and a New Gene: Evidence for a Novel Complex in ER-to-Golgi Transport

Genetics ◽

10.1093/genetics/149.2.833 ◽

1998 ◽

Vol 149 (2) ◽

pp. 833-841

Author(s):

Yu Jiang ◽

Al Scarpa ◽

Li Zhang ◽

Shelly Stone ◽

Ed Feliciano ◽

...

Keyword(s):

Growth Defect ◽

Carboxypeptidase Y ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Specific Suppression ◽

Golgi Transport ◽

New Gene ◽

Insight Into ◽

Suppressor Screen

Abstract The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex. To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34°. This high copy suppressor screen resulted in the isolation of a new gene, called BET5. BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants. This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex. Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone α-factor at 37°. The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus. High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1). These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport. The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

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Synaptojanin family members are implicated in endocytic membrane traffic in yeast

Journal of Cell Science ◽

10.1242/jcs.111.22.3347 ◽

1998 ◽

Vol 111 (22) ◽

pp. 3347-3356 ◽

Cited By ~ 2

Author(s):

B. Singer-Kruger ◽

Y. Nemoto ◽

L. Daniell ◽

S. Ferro-Novick ◽

P. De Camilli

Keyword(s):

Synaptic Vesicles ◽

Direct Evidence ◽

Family Members ◽

Fluid Phase ◽

Growth Defect ◽

Temperature Sensitive ◽

Close Link ◽

Fluid Phase Endocytosis ◽

Synaptojanin 1

The synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

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