scholarly journals Transcription closed and open complex formation coordinate expression of genes with a shared promoter region

2019 ◽  
Vol 16 (161) ◽  
pp. 20190507
Author(s):  
Antti Häkkinen ◽  
Samuel M. D. Oliveira ◽  
Ramakanth Neeli-Venkata ◽  
Andre S. Ribeiro
Keyword(s):  
Transcription Initiation ◽  
Regulatory Elements ◽  
Promoter Elements ◽  
E Coli ◽  
Rate Limiting Step ◽  
Expression Of Genes ◽  
Active Transcription ◽  
Rate Limiting ◽  
Kinetics Of

Many genes are spaced closely, allowing coordination without explicit control through shared regulatory elements and molecular interactions. We study the dynamics of a stochastic model of a gene-pair in a head-to-head configuration, sharing promoter elements, which accounts for the rate-limiting steps in transcription initiation. We find that only in specific regions of the parameter space of the rate-limiting steps is orderly coexpression exhibited, suggesting that successful cooperation between closely spaced genes requires the coevolution of compatible rate-limiting step configuration. The model predictions are validated using in vivo single-cell, single-RNA measurements of the dynamics of pairs of genes sharing promoter elements. Our results suggest that, in E. coli , the kinetics of the rate-limiting steps in active transcription can play a central role in shaping the dynamics of gene-pairs sharing promoter elements.

scholarly journals Transcription closed and open complex formation coordinate expression of genes with a shared promoter region

2019 ◽  
Author(s):  
Antti Häkkinen ◽  
Samuel M. D. Oliveira ◽  
Ramakanth Neeli-Venkata ◽  
Andre S. Ribeiro
Keyword(s):  
Transcription Initiation ◽  
Regulatory Elements ◽  
Promoter Elements ◽  
E Coli ◽  
Rate Limiting Step ◽  
Expression Of Genes ◽  
Active Transcription ◽  
Rate Limiting ◽  
Kinetics Of

Many genes are spaced closely, allowing coordination without explicit control through shared regulatory elements and molecular interactions. We study the dynamics of a stochastic model of a gene-pair in a head-to-head configuration, sharing promoter elements, which accounts for the rate-limiting steps in transcription initiation. We find that only in specific regions of the parameter space of the rate-limiting steps is orderly co-expression exhibited, suggesting that successful cooperation between closely spaced genes requires the co-evolution of compatible rate-limiting step configuration. The model predictions are validated by in vivo single-cell, single-RNA measurements of the dynamics of pairs of genes sharing promoter elements. Our results suggest that, in E. coli, the kinetics of the rate-limiting steps in active transcription can play a central role in the dynamics of pairs of genes sharing promoter elements.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

scholarly journals Comparison of promoter-specific events during transcription initiation in mycobacteria

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 1942-1952 ◽  
Author(s):  
Arnab China ◽  
Priyanka Tare ◽  
Valakunja Nagaraja
Keyword(s):  
Protein Interactions ◽  
Transcription Initiation ◽  
Kinetic Properties ◽  
Kinetic Rate ◽  
Rate Limiting Step ◽  
Equilibrium Binding ◽  
Transcription Process ◽  
Kinetic Rate Constants ◽  
Rate Limiting ◽  
Kinetics Of

DNA–protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter–RNAP interactions during transcription initiation in the σ A-dependent promoters P rrnAPCL1 , P rrnB and P gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

scholarly journals ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions

2016 ◽  
Author(s):  
Haythem Latif ◽  
Stephen Federowicz ◽  
Ali Ebrahim ◽  
Janna Tarasova ◽  
Richard Szubin ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Receptor Protein ◽  
Elongation Complex ◽  
Template Strand ◽  
Rate Limiting Step ◽  
Dna Motif ◽  
Genome Scale ◽  
Rate Limiting

ABSTRACTNumerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.

scholarly journals Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

2016 ◽  
Vol 113 (43) ◽  
pp. E6562-E6571 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Magnetic Tweezers ◽  
Nucleoside Triphosphate ◽  
Biologically Relevant ◽  
Rate Limiting Step ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP–GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused–backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

scholarly journals The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation

1976 ◽  
Vol 155 (2) ◽  
pp. 331-344 ◽  
Author(s):  
B Robson ◽  
R H Pain
Keyword(s):  
Optical Rotation ◽  
Folding Pathway ◽  
Structural Elements ◽  
Native State ◽  
Guanidinium Chloride ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Unfolding Kinetics ◽  
Kinetics Of

1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with ‘native format’ [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.

scholarly journals In vivo kinetics of transcription initiation of the lar promoter in Escherichia coli. Evidence for a sequential mechanism with two rate-limiting steps

2011 ◽  
Vol 5 (1) ◽  
pp. 149 ◽  
Author(s):  
Meenakshisundaram Kandhavelu ◽  
Henrik Mannerström ◽  
Abhishekh Gupta ◽  
Antti Häkkinen ◽  
Jason Lloyd-Price ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
In Vivo Kinetics ◽  
Sequential Mechanism ◽  
Rate Limiting ◽  
Kinetics Of

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Kinetics of cyclization of S-ethoxycarbonylmethylisothiouronium chloride to 2-imino-4-thiazolidone

1979 ◽  
Vol 44 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Vladimír Macháček ◽  
Said A. El-bahai ◽  
Vojeslav Štěrba
Keyword(s):  
Hydrochloric Acid ◽  
Dilute Hydrochloric Acid ◽  
Base Catalysis ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Rate Limiting ◽  
Kinetics Of

Kinetics of formation of 2-imino-4-thiazolidone from S-ethoxycarbonylmethylisothiouronium chloride has been studied in aqueous buffers and dilute hydrochloric acid. The reaction is subject to general base catalysis, the β value being 0.65. Its rate limiting step consists in acid-catalyzed splitting off of ethoxide ion from dipolar tetrahedral intermediate. At pH < 2 formation of this intermediate becomes rate-limiting; rate constant of its formation is 2 . 104 s-1.

Kinetics and mechanism of cyclization of N-(2-methoxycarbonylphenyl)-N’-methylsulphonamide to 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide

1991 ◽  
Vol 56 (8) ◽  
pp. 1701-1710 ◽  
Author(s):  
Jaromír Kaválek ◽  
Vladimír Macháček ◽  
Miloš Sedlák ◽  
Vojeslav Štěrba
Keyword(s):  
Potassium Hydroxide ◽  
Acid Catalysis ◽  
Potassium Hydroxide Solution ◽  
Hydroxide Solution ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cyclization Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The cyclization kinetics of N-(2-methylcarbonylphenyl)-N’-methylsulfonamide (IIb) into 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (Ib) has been studied in ethanolamine, morpholine, and butylamine buffers and in potassium hydroxide solution. The cyclization is subject to general base and general acid catalysis. The value of the Bronsted coefficient β is about 0.1, which indicates that splitting off of the proton from negatively charged tetrahedral intermediate represents the rate-limiting and thermodynamically favourable step. In the solutions of potassium hydroxide the cyclization of dianion of the starting ester IIb probably becomes the rate-limiting step.

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