scholarly journals Abstract P-23: Histone N-terminal Tails Reduce Early Nucleosomal Pausing during Transcription

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S21-S21
Author(s):  
Nadezhda Gerasimova ◽  
Fu-Kai Hsieh ◽  
Vasily Studitsky
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Proximal Part ◽  
Histone Tails ◽  
E Coli ◽  
In Vivo Experiments ◽  
Nucleosomal Dna ◽  
Transcript Elongation

Background: Nucleosomes are the barriers to transcript elongation by RNA polymerase 2 (Pol 2) in vitro and in vivo. Formation and overcoming the barrier are important for transcription regulation. N-terminal tails of core histones do not affect the inner structure of nucleosomal core. However, strongly positively charged tails can interact with the DNA, thereby impeding polymerase progression through the template. Removal of histone tails was shown to facilitate transcription through a nucleosome by both yeast and human Pol 2, and the effect was most noticeable at lower ionic strength (40 mM KCl). In vivo experiments established a new mechanism of overcoming of +1 nucleosomal barrier by removal of histone tails by specific regulative proteinase. As +1 nucleosomal barrier is formed mostly by the promoter-proximal part of the nucleosomal DNA, here we address the effects of histone tails on elongation through this part of the nucleosome. Methods: We have studied the effect of histone tails on transcription by yeast Pol 2 and model enzyme E. coli RNA polymerase utilizing very similar mechanisms of elongation through chromatin. 603 nucleosomes were transcribed in vitro using purified proteins and components. To focus on the proximal part of the nucleosome, transcript elongation was conducted for a limited time and at low ionic strength. Results: For the phosphorylated form of yeast Pol 2 and E. coli RNAP, histone tail removal significantly reduces the strong nucleosome-specific pausing that the yeast polymerase encounters ∼15 bp within the 603 nucleosome and further downstream, leading to both increased traversal of the pause and the accumulation of complexes paused at more distal locations. However, tail removal did not lead to a significant increase in full traversal of either nucleosomal template. The effect of histone tails removal was cognate for both enzymes but differs in detailed effect on the barrier. Conclusion: Histone tails provide a significant part of the nucleosomal barrier to transcript elongation by Pol 2-type mechanism. The effect is very pronounced in the promoter-proximal part of the nucleosomal DNA, suggesting that histone tails could play a role during the regulation of the +1 nucleosomal barrier. The role of Pol 2 CTD phosphorylation and formation of the intranucleosomal loops in the regulation of +1 nucleosomal barrier will also be addressed.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II

1992 ◽  
Vol 12 (9) ◽  
pp. 4142-4152
Author(s):  
J Archambault ◽  
F Lacroute ◽  
A Ruet ◽  
J D Friesen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structural Integrity ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Chemical Mutagenesis ◽  
Yeast Genome ◽  
Transcript Elongation

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

scholarly journals FCP1, a Phosphatase Specific for the Heptapeptide Repeat of the Largest Subunit of RNA Polymerase II, Stimulates Transcription Elongation

2002 ◽  
Vol 22 (21) ◽  
pp. 7543-7552 ◽  
Author(s):  
Subhrangsu S. Mandal ◽  
Helen Cho ◽  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Danny Reinberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Carboxy Terminal Domain ◽  
Transcription Factor Iif ◽  
Transcript Elongation ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.

scholarly journals Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations

2001 ◽  
Vol 183 (7) ◽  
pp. 2289-2297 ◽  
Author(s):  
Marco P. Cicero ◽  
Meghan M. Sharp ◽  
Carol A. Gross ◽  
Kenneth N. Kreuzer
Keyword(s):  
Rna Polymerase ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Positive Control ◽  
Plaque Morphology ◽  
Mutant Proteins ◽  
E Coli

ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. AmotA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. colipromoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

Potential Candidates against COVID-19 Targeting RNA-Dependent RNA Polymerase: A Comprehensive Review

2021 ◽  
Vol 22 ◽  
Author(s):  
Neetu Agrawal ◽  
Ahsas Goyal
Keyword(s):  
Rna Polymerase ◽  
In Silico ◽  
Host Cells ◽  
Rna Dependent Rna Polymerase ◽  
In Vivo Experiments ◽  
Contagious Nature ◽  
In Silico Studies ◽  
Viral Enzyme

: Due to the extremely contagious nature of SARS-COV-2, it presents a significant threat to humans worldwide. A plethora of studies are going on all over the world to discover the drug to fight SARS-COV-2. One of the most promising targets is RNA-dependent RNA polymerase (RdRp), responsible for viral RNA replication in host cells. Since RdRp is a viral enzyme with no host cell homologs, it allows the development of selective SARS-COV-2 RdRp inhibitors. A variety of studies used in silico approaches for virtual screening, molecular docking, and repurposing of already existing drugs and phytochemicals against SARS-COV-2 RdRp. This review focuses on collating compounds possessing the potential to inhibit SARS-COV-2 RdRp based on in silico studies to give medicinal chemists food for thought so that the existing drugs can be repurposed for the control and treatment of ongoing COVID-19 pandemic after performing in vitro and in vivo experiments.

scholarly journals Plasticity of the Pjunc Promoter of ISEc11, a New Insertion Sequence of the IS1111 Family

2006 ◽  
Vol 188 (13) ◽  
pp. 4681-4689 ◽  
Author(s):  
Gianni Prosseda ◽  
Maria Carmela Latella ◽  
Mariassunta Casalino ◽  
Mauro Nicoletti ◽  
Stefano Michienzi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Insertion Sequence ◽  
Functional Characterization ◽  
Virulence Plasmid ◽  
E Coli ◽  
Starting Point ◽  
Natural Variant ◽  
The Right

ABSTRACT We describe identification and functional characterization of ISEc11, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEc11 is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEc11 is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the IS1111 family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter (PjuncC) containing a −10 box in the interstitial sequence and a −35 box upstream of the right IR. A natural variant of ISEc11 (ISEc11p), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEc11p forms a junction promoter (PjuncP) which, despite carrying −10 and −35 boxes identical to those of PjuncC, exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of PjuncP is 20-fold higher than the activity of PjuncC. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

scholarly journals Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
Author(s):  
Umender K. Sharma ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Sigma Factors ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

scholarly journals Inhibition of Transcription in Staphylococcus aureus by a Primary Sigma Factor-Binding Polypeptide from Phage G1

2009 ◽  
Vol 191 (12) ◽  
pp. 3763-3771 ◽  
Author(s):  
Mohammed Dehbi ◽  
Gregory Moeck ◽  
Francis F. Arhin ◽  
Pascale Bauda ◽  
Dominique Bergeron ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Growth Inhibitory ◽  
Σ Factor ◽  
Transcription In Vitro

ABSTRACT The primary sigma factor of Staphylococcus aureus, σSA, regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with σSA both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of σSA that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-σ factor may occlude the −35 element recognition domain of σSA. As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of σSA to DNA and σSA-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

scholarly journals Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

1992 ◽  
Vol 12 (9) ◽  
pp. 4142-4152 ◽  
Author(s):  
J Archambault ◽  
F Lacroute ◽  
A Ruet ◽  
J D Friesen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structural Integrity ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Chemical Mutagenesis ◽  
Yeast Genome ◽  
Transcript Elongation

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

scholarly journals Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evgeniya Schastnaya ◽  
Zrinka Raguz Nakic ◽  
Christoph H. Gruber ◽  
Peter Francis Doubleday ◽  
Aarti Krishnan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate ◽  
Acetate Metabolism ◽  
Post Translational Modification ◽  
E Coli ◽  
In Vivo Experiments ◽  
Functional Relevance ◽  
Regulation Of Enzyme Activity

AbstractProtein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

scholarly journals Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

1998 ◽  
Vol 180 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Russell K. Karls ◽  
Timothy J. Donohue
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rhodobacter Sphaeroides ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Initiation Site ◽  
E Coli ◽  
Rna Polymerase Holoenzyme

ABSTRACT These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroidescytochrome c 2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterizedEscherichia coli heat shock (ς32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Eς32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the E. coliEς32 −35 consensus sequence (G34T) increasedcycA P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes,cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Eς38) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).

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