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Results for “negative elongation factor”

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PubMed2024

Pause Patrol: Negative Elongation Factor's Role in Promoter-Proximal Pausing and Beyond.

RNA polymerase (Pol) II is highly regulated to ensure appropriate gene expression. Early transcription elongation is associated with transient pausing of RNA Pol II in the promoter-proximal region. In multicellular organisms, this pausing is stabilized by the association of transcription elongation factors DRB-sensitivity inducing factor (DSIF) and Negative Elongation Factor (NELF). DSIF is a broadly conserved transcription elongation factor whereas NELF is mostly restricted to the metazoan lineage. Mounting evidence suggests that NELF association with RNA Pol II serves as checkpoint for either release into rapid and productive transcription elongation or premature termination at promoter-proximal pause sites. Here we summarize NELF's roles in promoter-proximal pausing, transcription termination, DNA repair, and signaling based on decades of cell biological, biochemical, and structural work and describe areas for future research.

Promoter Regions, Genetic↗

PubMed2025

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase (Pol) II is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here, we analyzed transcription in species across the tree of life. Unicellular eukaryotes display an accumulation of Pol II near transcription start sites, which we propose transitioned to the longer-lived, focused pause observed in metazoans. This transition coincided with the evolution of new subunits in the negative elongation factor (NELF) and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat-shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

RNA Polymerase II↗

PubMed1978

Paracrystalline arrays of protein-synthesis elongation factor Tu. Comparison with polymerized actin.

Homogeneous protein synthesis elongation factor Tu from Escherichia coli forms aggregates at high concentrations of ammonium sulfate which have a filamentous appearance in the light microscope. Electron microscopy of negatively stained preparations shows that these aggregates are paracrystalline, including three different forms. On the basis of analyses by optical diffraction, this polymorphism can be explained in terms of three different tubular foldings of the same basic two-dimensional surface lattice. This can be compared with that underlying the structure of actin filaments, thus providing a crucial test of the putative relationship between the elongation factor and actin [Rosenbusch, J. P. et al. (1976) J. Supramol. Struct. 5, 391-396]. The differences between the surface lattices, in conjunction with the negative results of sensitive immunochemical tests for possible cross-reactivities between the two proteins, suggest that any such relationship is very remote.

Actins↗

PubMed1979

Polymerization of the bacterial elongation factor for protein synthesis, EF-Tu.

The bacterial elongation factor for protein synthesis, EF-Tu, polymerizes into fibrils at pH 6.0. These fibrils are 0.7 microM in diameter, at least 200 microns in length, and are positively birefringent. Electron microscopic observations of negatively stained images demonstrates that the EF-Tu fibrils consist of bundles of individual filaments, approximately 5nm in diameter, aligned parallel to the long axis of the fibril. Polymerized EF-Tu exchanges nucleotide rapidly and interacts with the other elongation factor, EF-Ts. The antibiotic kirromycin induces the polymerization of EF-Tu into fibrils and even larger structures under nonpolymerizing conditions.

Guanosine Diphosphate↗

PubMed2026

Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon.

Conjugative plasmids drive bacterial evolution and antibiotic resistance spread, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that Sfx and H-NS share similar DNA motifs and a preference for negative supercoiling, but occupy reciprocal genomic niches; Sfx is enriched on the R6K vir operon despite weak chromosomal binding, whereas H-NS displays the opposite preference. We show that Sfx binding to vir DNA critically depends on DNA topology and hypothesize that its selective targeting to R6K is mediated by Sfx-vir interactions and phase separation. Our results suggest that Sfx phase separates with R6K to ensure its preferential recruitment to the plasmid DNA and forms stable bridged nucleoprotein filaments that are impermeable to competitors such as H-NS. These findings reveal how histone-like proteins can partition the genome into distinct regulatory niches, a strategy likely mirrored across all life.

Operon↗

PubMed1977

Ternary complex formation between elongation factor Tu, GTP and aminoacyl-tRNA: an equilibrium study.

The equilibria between the elongation factor Tu-GTP complex (EF-Tu-GTP) from Escherichia coli and tyrosyl-tRNATyr from E. coli as well as phenylalanyl-tRNAPhe and seryl-tRNASer from yeast were studied using a novel procedure, which takes advantage of the protective effect of ternary complex formation on the stability of theaminoacyl bond against non-enzymatic hydrolysis. At 25 degrees C and at pH 7.4 tyrosyl-tRNATyr, phenylalanyl-tRNAPhe and seryl-tRNASer are bound with binding constants of 0.7 X 10(7) M-1, 5.0 X 10(7) M-1 and 0.5 X 10(7) M-1 respectively. The binding of aminoacyl-tRNA to EF-Tu-GTP has a negative deltaH of the order of 10 kcal/mol (42 kJ/mol). Complex formation is dependent on ionic strength: with 0.1 M KCl Kass = 0.8 X 10(7) M-1, with 0.5 M KCl Kass = 0.2 X 10(7) M-1 was determined for the binding of Tyr-tRNATyr.

Calorimetry↗

PubMed1978

Heat-stable enterotoxin produced by Shigella flexneri.

Filtrates and ultrasonics extracts of Shigella flexneri showed rapid permeability factor (PF) test and proved positive in suckling mice and ligated rabbit loop tests within 4 hr. Delayed PF was not detected and the rabbit loop dilatation test read after 18 to 24 hr, the mouse pad oedema reaction, the test for elongation effect of CHO cells were also negative. In the delayed PF test a strong "blanching" effect was observed. A filtrate of an Ent-Escherichia coli strain was positive only in the rapid PF test, while filtrate and ultrasonic extract prepared from the Ent+ E. coli strain showed a positive reaction in all tests for enterotoxins (ST and LT) including the rapid PF test. Ultrasonic extracts of a S. flexneri and an Ent- E. coli strain concentrated by freeze-drying were fractionated on Sephadex G-100 column. S. flexneri fractions of 60--70ml were positive for rapid PF, dilation capacity in suckling mice, and the blanching effect in the delayed PF test. No positive reaction was found in the delayed PF test and in CHO cell culture. Similar fractions of Ent- E. Coli carried substances responsible for the rapid PF and the blanching effect, but without suckling mice positivity.

Bacterial Toxins↗

PubMed1976

Longitudinal tension of anterior tibial artery segments during constrictor responses.

Longitudinal tensions were recorded from in situ isolated segments of dog anterior tibial arteries during vasoconstrictor responses. Two factors appeared to be responsible for longitudinal tensions occurring during vasoconstrictor responses. One was due to changes in intraluminal pressure and appeared to be the dominant factor. Increases in intraluminal pressures produced decreases in longitudinal tensions, indicating that the vessel segments were elongated. Thus, increases in intraluminal pressure passively stretched the blood vessel walls so that increases in both the circumferential and longitudinal axes of the vessel segment occurred. These findings agree with similar findings reported earlier (4, 7, 8). The other factor was apparently due to changes in longitudinal tension produced by vascular smooth muscle contraction. In some cases longitudinal tension changes were positive, in others they were negative. Increases in longitudinal tension were probably the result of contractions of helical smooth muscle elements. Decreases in tension may have been due to displacement of tissue parallel to the long axis of the vessel as smooth muscle elements contracted.

Animals↗

PubMed1978

[Poly (ADP-ribose), ADP-ribosylation of proteins and regulation of cell activity].

The nature of a before unknown biological activity of NAD as a substrate in protein modification reaction is considered. Upon enzymatic digestion of NAD its adenosinediphosphate ribose (ADPR) part is transferred to acceptor proteins. ADPR in its mono- or polymeric form is covalently linked to proteins at the expense of NAD's high energy bound. Negatively charged ADPR, in association with a protein, is able to alter the charge, conformation and biological activity of the latter. The reaction is important in structural rearrangements of chromatin, in the synthesis and repair of DNA, in cell growth and differentiation and in the mechanisms of actions of actions of bacterial toxins.

Animals↗

PubMed1975

2-phenylethanol and some of its amphiphilic derivatives as inhibitors of platelet aggregation. Structure-activity relationship.

The relationship between chemical structure and inhibitory activity of some simple water soluble phenylalkyl derivatives has been investigated. The inhibitory effect of the following analogous and homologous derivatives increases in the sequence: phenylacetic acid less than 2-phenylethanol less than 2-phenylethylamine = 4-phenylbutyric acid less than 3-phenylpropanol less than 4-phenylbutylamine, i.e., the inhibitory effect is potentiated. 1. by elongation of the alkyl chain, which is accompanied by an increase of the lipophilic character, and 2. when the polar portion is represented by the positively charged amino-group. The effect of these amphiphilic compounds on platelet aggregation appears to be mediated through polar and apolar interactions with the outer layers of the plasma membrane. The fact that the amino derivatives are the most active inhibitors suggests that negatively charged groups may be involved in the polar binding of these inhibitors. Experiments with neuraminidase-treated platelets revealed that the enzymatically removable sialic acid residues are not those negatively charged binding sites. Phentolamine and propranolol which block adrenergic alpha- and beta-receptors, respectively, do not influence the inhibitory effect of these agents.

Adenosine Diphosphate↗

PubMed1976

Demonstration of the existence of a single morphological type of gonadotrophic cell in Ellobius lutescens (Microtinae) by an ultrastructural analysis of their development under various physiological and experimental conditions.

Ultrastructural studies of pituitaries from Ellobius lutescens (immature males and females, adult hypogonadic males, and virgin and pregnant females) show that the gonadotrophic cells are characterized by a lamellar or vacuolar rough endoplasmic reticulum (RER), a spirally-arranged Golgi apparatus, elongated mitochondria and secretory granules of variable density and size (150-500 mmu). Ultrastructural differences between gonadotrophic cells previously determined by light microscopy correspond to changes in the development of the protein synthetic apparatus and in the intensity of hormonal discharge. Type 2 gonadotrophs always appear to be more active than type 1 gonadotrophs. After castration, all gonadotrophic cells develop into the same form of "castration cell", although type 1 gonadotrophs change more slowly than type 2 cells. Treatment with testosterone induces an inverse development of the gonadotrophic cells which take on the appearance of resting cells similar to those found in immature animals, where the two cell types are also identical. Thus, only one morphological type of gonadotrophic cell can be identified in Ellobius lutescens. Moreover, the gonadotrophic cells of the hypogonadic adult male have the same appearnance as those of the female two months after castration, whic proves that the negative feedback mechanism which regulates gonadtrophic function is defective in adult male Ellobius lutescens.

Animals↗