scholarly journals Chemical roadblocking of DNA transcription for nascent RNA display

2020 ◽  
Vol 295 (19) ◽  
pp. 6401-6412
Author(s):  
Eric J. Strobel ◽  
John T. Lis ◽  
Julius B. Lucks
Keyword(s):  
Escherichia Coli ◽  
Rna Structure ◽  
Triethylene Glycol ◽  
Site Specific ◽  
Rna Molecules ◽  
Protein Factor ◽  
Nascent Rna ◽  
Dna Strand ◽  
And Function

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2′-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

scholarly journals Chemical transcription roadblocking for nascent RNA display

2019 ◽  
Author(s):  
Eric J. Strobel ◽  
John T. Lis ◽  
Julius B. Lucks
Keyword(s):  
Rna Polymerase ◽  
Rna Structure ◽  
Triethylene Glycol ◽  
Rna Molecules ◽  
Protein Factor ◽  
Nascent Rna ◽  
Dna Strand ◽  
And Function ◽  
Dna Template

AbstractSite-specific arrest of RNA polymerase is fundamental to several technologies that measure RNA structure and function. Current in vitro transcription ‘roadblocking’ approaches inhibit transcription elongation using a protein blockade bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires the inclusion of a protein factor that is extrinsic to the minimal in vitro transcription reaction. In this work, we show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification efficiently and stably halts Escherichia coli RNA polymerase transcription. To facilitate diverse applications of chemical transcription roadblocking, we establish a simple and sequence-independent method for the preparation of internally modified double-stranded DNA templates by sequential PCR and translesion synthesis. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables nascent RNA molecules to be displayed from RNA polymerase in a minimal in vitro transcription reaction.

scholarly journals Histone carbonylation in vivo and in vitro

2000 ◽  
Vol 351 (3) ◽  
pp. 769-777 ◽  
Author(s):  
Georg T. WONDRAK ◽  
Daniel CERVANTES-LAUREAN ◽  
Elaine L. JACOBSON ◽  
Myron K. JACOBSON
Keyword(s):  
Histone H1 ◽  
Nuclear Proteins ◽  
Carbonyl Groups ◽  
Cell Nuclei ◽  
Strand Breaks ◽  
Core Histones ◽  
Dna Strand ◽  
And Function

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H2O2, but was significantly increased following alkylating stress induced by N-methyl-N´-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.

The influence of radiomimetic substances on deoxyribonucleic acid synthesis and function studied in Escherichia coli / phage systems - III. Mutation of T 2 bacteriophage as a consequence of alkylation in vitro : the uniqueness of ethylation

1959 ◽  
Vol 150 (941) ◽  
pp. 497-508 ◽  
Keyword(s):  
Escherichia Coli ◽  
Burst Size ◽  
Alkylating Agents ◽  
Direct Consequence ◽  
Acid Synthesis ◽  
Deoxyribonucleic Acid Synthesis ◽  
Methane Sulphonate ◽  
Average Burst Size ◽  
And Function

Of a comprehensive set of alkylating agents tested, only two, namely, ethyl methane sulphonate and diethyl sulphate, have been found so to interact with T 2 bacteriophage that cells of Escherichia coli , infected with phage treated extracellularly, manifest a considerably increased likelihood of yielding mutated phage. Since this increase can occur where the infective titre of the phage and the latent period and average burst size of the infected bacteria remain unchanged, it is considered that the increased mutation rate is a direct consequence of the chemical treatment, although the alkylation itself does not constitute the mutation. A study of the manner of inactivation of the phage by these agents has not revealed any characteristic difference between ethylation and other alkylations which could be held to account for its apparent uniqueness.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

Simple topology: FtsK-directed recombination at the dif site

2013 ◽  
Vol 41 (2) ◽  
pp. 595-600 ◽  
Author(s):  
Ian Grainge
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Motor Domain ◽  
Site Specific ◽  
Multifunctional Protein ◽  
Supercoiled Plasmid ◽  
C Terminus ◽  
Dna Translocase

FtsK is a multifunctional protein, which, in Escherichia coli, co-ordinates the essential functions of cell division, DNA unlinking and chromosome segregation. Its C-terminus is a DNA translocase, the fastest yet characterized, which acts as a septum-localized DNA pump. FtsK's C-terminus also interacts with the XerCD site-specific recombinases which act at the dif site, located in the terminus region. The motor domain of FtsK is an active translocase in vitro, and, when incubated with XerCD and a supercoiled plasmid containing two dif sites, recombination occurs to give unlinked circular products. Despite years of research the mechanism for this novel form of topological filter remains unknown.

scholarly journals The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

2003 ◽  
Vol 185 (10) ◽  
pp. 3076-3080 ◽  
Author(s):  
Dominic Esposito ◽  
Gary F. Gerard
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Essential Role ◽  
Bacteriophage Λ ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
In Vivo Recombination

ABSTRACT The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

S1 mapping of purified nascent transcripts of simian virus 40

1984 ◽  
Vol 4 (4) ◽  
pp. 625-633
Author(s):  
D E Lycan ◽  
K J Danna
Keyword(s):  
Affinity Chromatography ◽  
Salt Concentration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Rna Molecules ◽  
S1 Mapping ◽  
Nascent Rna ◽  
Lower Temperature ◽  
Nascent Transcripts

We purified nascent simian virus 40 late transcripts by incubating viral transcriptional complexes, isolated from infected BSC-1 cells, in a reaction mixture that contained mercurated CTP; RNA molecules that had incorporated mercurated residues in vitro were isolated by sulfhydrylcellulose affinity chromatography. The nascent RNA was hybridized to an end-labeled HindIII C probe fragment (0.646 to 0.86 map unit), and the hybrids were analyzed by S1 mapping. Most of the products of digestion corresponded to unspliced transcripts with 5' ends mapping at nucleotides 325, 260, and 195, which are positions of the 5' ends of mature, cytoplasmic late mRNA species. In addition, two minor products diagnostic of splicing at the acceptor junctions mapping at nucleotides 556 and 443 were detected. Because the abundance of these products was not diminished by repurifying the nascent RNA through a second round of sulfhydrylcellulose chromatography, these products did not originate from contaminating non-nascent RNA. Moreover, the generation of these products was not affected when a higher salt concentration and lower temperature were used for S1 digestion, conditions that should decrease artifactual cleavage by S1 in A + U-rich regions of colinear hybrids. Therefore, it is likely that some simian virus 40 RNA chains are spliced before release from the template.

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