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At least 19 recordsLinked to original sources
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↗
PubMed1992

Inhibition of bacterial protein synthesis by elongation-factor-Tu-binding antibiotics MDL 62,879 and efrotomycin.

MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis. MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu). We prepared ribosomes and protein-synthesis elongation factors from three sources: E. coli, Bacillus subtilis, and a strain of B. subtilis selected for resistance to MDL 62,879 (strain G1674). Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B. subtilis and E. coli. We showed that in cell-free protein synthesis: (a) E. coli was sensitive to both MDL 62,879 and efrotomycin; (b) B. subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B. subtilis G1674 was resistant to both antibiotics. In the E. coli system and in the system from wild-type B. subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B. subtilis G1674. This demonstrates that the antibiotic acts by inhibition of EF-Tu. In contrast, extracts from B. subtilis failed to restore activity in an efrotomycin-inhibited E. coli system. Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action.

Anti-Bacterial Agents↗
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↗
PubMed1992

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.

Amino Acid Sequence↗
PubMed1992

The sequence of the gene encoding elongation factor Tu from Chlamydia trachomatis compared with those of other organisms.

Nucleotide (nt) sequences encoding the elongation factor Tu (EF-Tu), tRNA(Thr) and tRNA(Trp) from Chlamydia trachomatis have been determined. The environment of the EF-Tu-encoding gene (tuf), between two tRNA gene sequences, suggests that it is part of a tufB locus. The nt sequence and the deduced amino acid (aa) sequence were aligned with comparable sequences from other organisms and the resulting data bases were used to infer phylogenies. Phylogenetic trees based on aa sequences and nt sequences are similar, but not completely congruent with rRNA gene-based phylogenies. Both the nt and aa sequence trees concur on the early divergence of Thermotoga and Chlamydia from the bacterial root. The aa alignment highlights the presence of four unique Cys residues in the chlamydial sequence which are found at strictly conserved positions in other sequences. Further peculiarities of the chlamydial and eubacterial sequences have been mapped to the X-ray crystallographic structure of the protein.

Amino Acid Sequence↗
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↗
PubMed1992

Regulation of c-myc transcription initiation and elongation.

The c-myc protooncogene plays an important role in the regulation of cellular proliferation and differentiation. Selective downregulation of c-myc expression induces differentiation of leukemic cells, whereas constitutive overexpression prevents differentiation of the same cell types. The c-myc gene is comprised of three exons, the first of which is largely untranslated. Both c-myc mRNA and the c-myc gene product have very short half-lives, resulting in a very sensitive regulation of this important gene. Both transcriptional and posttranscriptional regulation of the c-myc gene expression have been documented. Transcriptional regulation can occur at the level of transcriptional initiation or elongation. A number of c-myc promoter-binding proteins have been identified and characterized. These include positive and negative regulatory factors. It appears that the c-myc gene is very tightly regulated in response to positive and negative growth stimuli.

Animals↗
PubMed1992

The organization and expression of essential transcription translation component genes in the extremely thermophilic eubacterium Thermotoga maritima.

A 5789-nucleotide-long EcoRI fragment from the genome of Thermotoga maritima, identified by cross-hybridization to L11, L1, L10, and L12 ribosomal protein gene sequences from Escherichia coli, was cloned and sequenced. The fragment encodes five tRNAs (tRNA(met1), anticodon complementary to AUG; tRNA(met2), AUG; tRNA(thr), ACA; tRNA(tyr), UAC; tRNA(trp), UGG), the transcription termination-antitermination factor nusG, the four 50 S subunit ribosomal proteins L11, L1, L10, and L12, and the amino-terminal portion of the RNA polymerase beta subunit protein. The five tRNA genes, the nusG gene, and the L11, L1, L10, and L12 ribosomal protein genes form a complex transcription unit. Transcripts appear to be initiated from an upstream promoter, P1, located in front of the tRNA(met1) gene and from three internal promoters: P2 is located immediately in front of the tRNA(met2) gene; PL10 is near the beginning of the L1-L10 intergenic space, and PL12 is at the end of the L10 gene sequence. The tRNA sequences are excised from the leader regions of the P1- and P2-initiated transcripts. Three putative but potentially important regulatory sequences were identified within this operon: an L1 translational control site, a transcription attenuator, and a strong rho-independent terminator. The strong terminator located distal to the L12 gene overlaps a fifth promoter, P beta, which is used to initiate transcripts of the downstream RNA polymerase beta subunit gene. The T. maritima NusG protein exhibits 43% amino acid sequence identity when aligned to the E. coli protein; the alignment is interrupted by a large 171-amino acid-long insertion into the T. maritima protein after codon 45.

Amino Acid Sequence↗
PubMed1992

Biopolymers from marine invertebrates. XIII. Characterization of an antibacterial protein, dolabellanin A, from the albumen gland of the sea hare, Dolabella auricularia.

An antibacterial factor, dolabellanin A, was purified from the albumen gland of a sea hare, Dolabella auricularia. Purified dolabellanin A was a glycoprotein of 250 kilodaltons consisting of 4 subunits, and showed both antibacterial and antineoplastic activities. The two activities were lost in parallel on heating and at low and high pH. This factor was half-maximally active for gram-positive and -negative bacteria at 0.018-0.48 microgram/ml, and its action was not bactericidal but bacteriostatic. Dolabellanin A did not induce morphological elongation of bacteria or the release of adenosine triphosphate, but it completely inhibited the syntheses of deoxyribonucleic acid (DNA) and ribonucleic acid by E. coli within 6 min. These results suggest that dolabellanin A, which is found in a marine invertebrate, the sea hare, is a new antibacterial protein, and that it exerts its action by inhibiting nucleic acid synthesis, as does a DNA-inhibiting chemotherapeutic drug.

Amino Acid Sequence↗
PubMed1992

Apparent association constants of tRNAs for the ribosomal A, P, and E sites.

Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.

Acylation↗
PubMed1992

Epithelioid and spindle-cell hemangioendothelioma of the spleen. Report of a distinctive splenic vascular neoplasm of childhood.

A case of a distinctive vascular neoplasm of the spleen in a 3-year-old boy is described. The tumor was characterized histologically by a biphasic growth pattern, with discrete nodular areas composed of atypical round, epithelioid cells with large nuclei and prominent nucleoli, and areas showing an intricate proliferation of vascular channels lined by elongated spindle cells. Immunohistochemical studies showed cytoplasmic staining of the tumor cells with factor VIII-related antigen, Ulex europaeus lectin, and vimentin antibodies. Stains for keratin, actin, desmin, lysozyme, and S-100 protein were negative in the tumor cells. Electron microscopy revealed a fairly cohesive population of cells that contained mature and immature cell junctions, basal lamina material, and surface pinocytotic activity consistent with vascular endothelial cells. Five-year follow-up has shown the patient to be alive and free of disease. This case appears to represent a previously unreported primary vascular neoplasm of the spleen showing combined features of epithelioid and spindle-cell hemangioendothelioma. The lesion should be distinguished from other benign and malignant vascular proliferations of the spleen such as Kaposi's sarcoma, angiosarcoma, and the recently described littoral-cell angioma.

Child, Preschool↗
PubMed1992

Identification of nuclear factors which interact with the 5' flanking region of the EF-1 alpha O gene in Xenopus laevis.

The EF-1 alpha O gene of Xenopus laevis is a stage-specific gene, being transcribed in oogonia and oocytes, but not in postmeiotic germ cells and terminally differentiated cells. We found that two trans-acting factors from oocyte nuclear extract are able to interact with a DNA sequence in the 5'-upstream region of the EF-1 alpha O gene. Methylation interference experiments suggested that the two factors recognised the same DNA element. Gel retardation assays indicated that part of the protein binding site could be confined to a 21 bp sequence, located between -51 and -72, relative to the cap site. Interestingly, this region shares great homology to a negative regulatory segment in the promoter of the TFIIIA gene, another developmentally regulated gene.

Animals↗
PubMed1992

Salmonella cytotonic and cytolytic factors: their detection in Chinese hamster ovary cells and antigenic relatedness.

Thirty nine strains of Salmonella belonging to 14 different serotypes were screened for the production of cytotonic and cytolytic factors by assayed Chinese hamster ovary (CHO) cells. The cell-free culture supernatants (CFCS) of 32 isolates caused elongation and increase in size of CHO cells while the CFCSs completely lysed the cells. The cytotonic effect in CHO cells correlated precisely with fluid the CFCSs completely lysed the cells. The cytotonic effect in CHO cells correlated precisely with fluid accumulation in the rabbit ligated ileal assay in that 24 isolates yielded positive results in both assays and 13 were found negative in both. Antiserum to S. typhimurium enterotoxin, but not that to cholera toxin or Shiga toxin, neutralized the cytotonic activity present in the CFCS and reacted with the latter in immunodiffusion and coagglutination tests. The cytolytic factor produced by two strains reacted neither with antiserum to Salmonella enterotoxin nor with that the Shiga toxin.

Agglutination Tests↗
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↗
PubMed1992

Effects of template topology on RNA polymerase pausing during in vitro transcription of the Escherichia coli rrnB leader region.

Transcription elongation catalysed by DNA-dependent RNA polymerase does not occur at a constant rate. Instead, during the transcription of many genes pausing occurs at defined template positions. Pausing is known to be influenced by the intracellular NTP concentration, the secondary structure of the growing transcript or by transcription factors like NusA. We have investigated the effects of the template topology of transcriptional pauses in the presence and absence on purified NusA protein. Taking advantage of a method for quantifying transcriptional pauses we have studied pausing behaviour during in vitro transcription of the early region of a plasmid-encoded ribosomal RNA operon. Plasmid templates with different superhelical densities (sigma between +0.0017 and -0.055) were employed in transcription elongation assays. If linearized or relaxed templates are used, some of the characteristic pauses can no longer be detected. For the stronger pauses we could demonstrate a direct correlation between pause strength and the negative superhelical densities of the templates used. This correlation is observed regardless of whether or not pauses are dependent upon NusA. Changes in the average transcription elongation rate, caused by variations in the NTP concentration or the temperature, do not appear to have a comparable effect on transcription pausing. The results are consistent with the assumption that the template topology has a regulatory function in transcription elongation of rRNA genes in Escherichia coli.

Bacterial Proteins↗