scholarly journals Regulation of rRNA Transcription Correlates with Nucleoside Triphosphate Sensing

2001 ◽  
Vol 183 (21) ◽  
pp. 6315-6323 ◽  
Author(s):  
Melanie M. Barker ◽  
Richard L. Gourse
Keyword(s):  
Growth Rate ◽  
Core Promoter ◽  
Nucleoside Triphosphate ◽  
Wild Type ◽  
Rate Dependent ◽  
Rrna Transcription ◽  
In Vivo Growth ◽  
Wild Type Promoter

ABSTRACT We have previously shown that the activity of theEscherichia coli rRNA promoter rrnB P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 differentrrnB P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The sevenrrnB P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the fis gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the rrnB P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which rrnB P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).

scholarly journals Regulation of rRNA Transcription Is Remarkably Robust: FIS Compensates for Altered Nucleoside Triphosphate Sensing by Mutant RNA Polymerases at Escherichia coli rrn P1 Promoters

2000 ◽  
Vol 182 (7) ◽  
pp. 1969-1977 ◽  
Author(s):  
Michael S. Bartlett ◽  
Tamas Gaal ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Transcription Initiation ◽  
Rrna Synthesis ◽  
Nucleoside Triphosphate ◽  
Wild Type ◽  
Core Promoters ◽  
Rate Dependent ◽  
Rrna Transcription

ABSTRACT We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP β′ Δ215–220 and β RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the rrntranscription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent K ATP for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.

scholarly journals Multiple Mechanisms Are Used for Growth Rate and Stringent Control of leuV Transcriptional Initiation inEscherichia coli

1999 ◽  
Vol 181 (18) ◽  
pp. 5771-5782 ◽  
Author(s):  
Dmitry K. Pokholok ◽  
Maria Redlak ◽  
Charles L. Turnbough ◽  
Sara Dylla ◽  
Walter M. Holmes
Keyword(s):  
Growth Rate ◽  
Binding Site ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Cellular Level ◽  
Structural Variants ◽  
Stringent Control ◽  
The Stability

ABSTRACT Expression of the Escherichia coli leuV operon, which contains three tRNA1 Leu genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3′,5′-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of theleuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those ofrrnBp 1. The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuVpromoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from theleuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp 1. Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

scholarly journals Human Immunodeficiency Virus Bearing a Disrupted Central DNA Flap Is Pathogenic In Vivo

2007 ◽  
Vol 81 (11) ◽  
pp. 6146-6150 ◽  
Author(s):  
Matthew D. Marsden ◽  
Jerome A. Zack
Keyword(s):  
Human Immunodeficiency Virus ◽  
Growth Characteristics ◽  
Deficient Mutant ◽  
Wild Type ◽  
Hiv Replication ◽  
Immunodeficiency Virus ◽  
In Vivo Growth ◽  
Kinetics Of

ABSTRACT The central DNA flap is an important component of lentiviral vectors, but its significance in the context of wild-type human immunodeficiency virus (HIV) is currently unclear. To address this issue, we have compared the in vitro infection kinetics of NL4-3 with those of a flap-deficient mutant and evaluated the in vivo growth characteristics of these viruses by using the SCID-hu mouse model of HIV infection. Flap-deficient virus was only modestly attenuated in vitro, as assessed by single-round and spreading infection assays, and exhibited levels of replication and pathogenesis close to those of the wild-type in vivo. Hence, an intact central flap is not essential for HIV replication.

scholarly journals Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

2004 ◽  
Vol 78 (12) ◽  
pp. 6091-6101 ◽  
Author(s):  
K. Sivakumaran ◽  
Seung-Kook Choi ◽  
Masarapu Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Core ◽  
Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

Production of tumours and roots by Ephedra following Agrobacterium rhizogenes infection

1994 ◽  
Vol 72 (2) ◽  
pp. 203-207 ◽  
Author(s):  
Niamh A. O'Dowd ◽  
David H. S. Richardson
Keyword(s):  
Growth Rate ◽  
Plant Growth ◽  
Agrobacterium Rhizogenes ◽  
Dry Weight ◽  
Wild Type ◽  
Bacterial Strains ◽  
Heat Treated ◽  
Slow Growing

This paper contains the first report that stems of the Gnetophyte Ephedra respond to infection by Agrobacterium rhizogenes by producing roots and tumours in vivo and in vitro. Of the bacterial strains employed, the wild-type Ar2629 gave the maximum response, and strain LBA9402 was also effective. In no case did heat-treated A. rhizogenes produce tumours or roots. Excised tumour tissues were cultured for more than 2 years in the absence of exogenous plant-growth regulators without any deterioration in growth rate. In vivo tumours of Ephedra fragilis and Ephedra minima contained up to 0.3% dry weight l-ephedrine, and slow-growing in vitro cultured tumours of E. fragilis contained up to 0.01% l-ephedrine, but alkaloid was not detected in faster growing isolates. Key words: Agrobacterium rhizogenes, alkaloid, Ephedra, l-ephedrine, Gnetophytes, gymnosperm, tumours.

scholarly journals Evidence that the Promoter Can Influence Assembly of Antitermination Complexes at Downstream RNA Sites

2006 ◽  
Vol 188 (6) ◽  
pp. 2222-2232 ◽  
Author(s):  
Ying Zhou ◽  
Ting Shi ◽  
Mark A. Mozola ◽  
Eric R. Olson ◽  
Karla Henthorn ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Wild Type ◽  
Present Evidence ◽  
N Protein ◽  
In Vivo Experiments ◽  
Phage Development ◽  
A Site ◽  
Wild Type Promoter

ABSTRACT The N protein of phage λ acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the λ early P R promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P R, at positions −14 and −45 (yielding P R-GA), reduces effectiveness of N modification, while an additional mutation at position −30 (yielding P R-GCA) suppresses this effect. In vivo, the level of P R-GA-directed transcription was twice as great as the wild-type level, while transcription directed by P R-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P R-GA in vitro was roughly one-third the rate for wild-type P R. We ascribe this apparent discrepancy to an effect of the mutations in P R-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.

scholarly journals Growth rate-dependent flexural rigidity of microtubules influences pattern formation in collective motion

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hang Zhou ◽  
Naoto Isozaki ◽  
Kazuya Fujimoto ◽  
Ryuji Yokokawa
Keyword(s):  
Growth Rate ◽  
Pattern Formation ◽  
Collective Motion ◽  
Flexural Rigidity ◽  
Biological Activities ◽  
Gliding Motility ◽  
Regulation Mechanism ◽  
Rate Dependent

Abstract Background Microtubules (MTs) are highly dynamic tubular cytoskeleton filaments that are essential for cellular morphology and intracellular transport. In vivo, the flexural rigidity of MTs can be dynamically regulated depending on their intracellular function. In the in vitro reconstructed MT-motor system, flexural rigidity affects MT gliding behaviors and trajectories. Despite the importance of flexural rigidity for both biological functions and in vitro applications, there is no clear interpretation of the regulation of MT flexural rigidity, and the results of many studies are contradictory. These discrepancies impede our understanding of the regulation of MT flexural rigidity, thereby challenging its precise manipulation. Results Here, plausible explanations for these discrepancies are provided and a new method to evaluate the MT rigidity is developed. Moreover, a new relationship of the dynamic and mechanic of MTs is revealed that MT flexural rigidity decreases through three phases with the growth rate increases, which offers a method of designing MT flexural rigidity by regulating its growth rate. To test the validity of this method, the gliding performances of MTs with different flexural rigidities polymerized at different growth rates are examined. The growth rate-dependent flexural rigidity of MTs is experimentally found to influence the pattern formation in collective motion using gliding motility assay, which is further validated using machine learning. Conclusion Our study establishes a robust quantitative method for measurement and design of MT flexural rigidity to study its influences on MT gliding assays, collective motion, and other biological activities in vitro. The new relationship about the growth rate and rigidity of MTs updates current concepts on the dynamics and mechanics of MTs and provides comparable data for investigating the regulation mechanism of MT rigidity in vivo in the future. Graphic Abstract

scholarly journals Regeneration linked miRNA modify tumor phenotype and can enforce multi-lineage growth arrest in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Siamak Salehi ◽  
Oliver D. Tavabie ◽  
Augusto Villanueva ◽  
Julie Watson ◽  
David Darling ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Growth Inhibition ◽  
Tumor Aggressiveness ◽  
Novel Treatment ◽  
Tumor Phenotype ◽  
Aggressive Tumor ◽  
In Vivo Growth ◽  
Regulation Of Regeneration

AbstractRegulated cell proliferation is an effector mechanism of regeneration, whilst dysregulated cell proliferation is a feature of cancer. We have previously identified microRNA (miRNA) that regulate successful and failed human liver regeneration. We hypothesized that these regulators may directly modify tumor behavior. Here we show that inhibition of miRNAs -503 and -23a, alone or in combination, enhances tumor proliferation in hepatocyte and non-hepatocyte derived cancers in vitro, driving more aggressive tumor behavior in vivo. Inhibition of miRNA-152 caused induction of DNMT1, site-specific methylation with associated changes in gene expression and in vitro and in vivo growth inhibition. Enforced changes in expression of two miRNA recapitulating changes observed in failed regeneration led to complete growth inhibition of multi-lineage cancers in vivo. Our results indicate that regulation of regeneration and tumor aggressiveness are concordant and that miRNA-based inhibitors of regeneration may constitute a novel treatment strategy for human cancers.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

2021 ◽  
pp. 1-24
Author(s):  
Juho-Matti Renko ◽  
Arun Kumar Mahato ◽  
Tanel Visnapuu ◽  
Konsta Valkonen ◽  
Mati Karelson ◽  
...  
Keyword(s):  
Mapk Signaling ◽  
Dopamine Neurons ◽  
Dopamine Depletion ◽  
Wild Type ◽  
Disease Modifying Therapy ◽  
Immortalized Cells ◽  
Midbrain Dopamine ◽  
6 Hydroxydopamine

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

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