Nus A protein affects transcriptional pausing and termination in vitro by binding to different sites on the transcription complex

Biochemistry ◽  
1988 ◽  
Vol 27 (15) ◽  
pp. 5622-5627 ◽  
Author(s):  
Curt D. Sigmund ◽  
Edward A. Morgan
Keyword(s):  
Transcriptional Pausing

scholarly journals Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

2004 ◽  
Vol 186 (16) ◽  
pp. 5392-5399 ◽  
Author(s):  
Frank J. Grundy ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Assay System ◽  
Nascent Transcript ◽  
Vitro Assay ◽  
T Box ◽  
Transcription Antitermination ◽  
Transcriptional Pausing ◽  
Transcription Complexes ◽  
Kinetics Of

ABSTRACT Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNAGly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

scholarly journals Transcriptional Pausing in the Bacillus subtilis pyr Operon In Vitro: a Role in Transcriptional Attenuation?

2003 ◽  
Vol 185 (16) ◽  
pp. 4764-4771 ◽  
Author(s):  
Hesheng Zhang ◽  
Robert L. Switzer
Keyword(s):  
Bacillus Subtilis ◽  
Rna Binding ◽  
Bacterial Species ◽  
Regulatory Protein ◽  
Stem Loop ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Transcriptional Pausing

ABSTRACT The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro. Using templates with each of the three known B. subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo. With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

The σ24 Subunit of Escherichia coli RNA Polymerase Can Induce Transcriptional Pausing in vitro

2019 ◽  
Vol 84 (4) ◽  
pp. 426-434
Author(s):  
A. B. Shikalov ◽  
D. M. Esyunina ◽  
D. V. Pupov ◽  
A. V. Kulbachinskiy ◽  
I. V. Petushkov
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcriptional Pausing

scholarly journals Stability of Drosophila RNA polymerase II elongation complexes in vitro.

1992 ◽  
Vol 12 (5) ◽  
pp. 2067-2077 ◽  
Author(s):  
D D Kephart ◽  
N F Marshall ◽  
D H Price
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Subsequent Treatment ◽  
Elongation Rate ◽  
Nucleoside Analog ◽  
Purine Nucleoside ◽  
Elongation Factors ◽  
Transcriptional Pausing

We show that nuclear extract from Drosophila Kc cells supports efficient elongation by RNA polymerase II initiated from the actin 5C promoter. The addition of 0.3% Sarkosyl, 1 mg of heparin per ml, or 250 mM KCl immediately after initiation has two effects. First, the elongation rate is reduced 80 to 90% as a result of the inhibition of elongation factors. Second, there is an increase in the amount of long runoff RNA, suggesting that there is an early block to elongation that is relieved by the disruptive reagents. Consistent with the first effect, we find that the ability of factor 5 (TFIIF) to stimulate the elongation rate is inhibited by the disruptive agents when assayed in a defined system containing pure RNA polymerase II and a dC-tailed template. The disruptive agents also inhibit the ability of DmS-II to suppress transcriptional pausing but only slightly reduce the ability of DmS-II to increase the elongation rate twofold. The pause sites encountered by RNA polymerase II after initiation at a promoter and subsequent treatment with the disruptive reagents are also recognized by pure polymerase transcribing a dC-tailed template. It has been suggested that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits RNA polymerase II during elongation, but we find that the purine nucleoside analog has no effect on elongation complexes containing RNA over 500 nucleotides in length or on the action of factor 5 or DmS-II in the defined system.

Stability of Drosophila RNA polymerase II elongation complexes in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2067-2077
Author(s):  
D D Kephart ◽  
N F Marshall ◽  
D H Price
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Subsequent Treatment ◽  
Elongation Rate ◽  
Nucleoside Analog ◽  
Purine Nucleoside ◽  
Elongation Factors ◽  
Transcriptional Pausing

We show that nuclear extract from Drosophila Kc cells supports efficient elongation by RNA polymerase II initiated from the actin 5C promoter. The addition of 0.3% Sarkosyl, 1 mg of heparin per ml, or 250 mM KCl immediately after initiation has two effects. First, the elongation rate is reduced 80 to 90% as a result of the inhibition of elongation factors. Second, there is an increase in the amount of long runoff RNA, suggesting that there is an early block to elongation that is relieved by the disruptive reagents. Consistent with the first effect, we find that the ability of factor 5 (TFIIF) to stimulate the elongation rate is inhibited by the disruptive agents when assayed in a defined system containing pure RNA polymerase II and a dC-tailed template. The disruptive agents also inhibit the ability of DmS-II to suppress transcriptional pausing but only slightly reduce the ability of DmS-II to increase the elongation rate twofold. The pause sites encountered by RNA polymerase II after initiation at a promoter and subsequent treatment with the disruptive reagents are also recognized by pure polymerase transcribing a dC-tailed template. It has been suggested that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits RNA polymerase II during elongation, but we find that the purine nucleoside analog has no effect on elongation complexes containing RNA over 500 nucleotides in length or on the action of factor 5 or DmS-II in the defined system.

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

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