scholarly journals Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays

2021 ◽  
Author(s):  
Eric J. Strobel
Keyword(s):  
Rna Polymerase ◽  
Magnetic Beads ◽  
Transcription Elongation ◽  
Biologically Active ◽  
Enzymatic Reactions ◽  
One Pot ◽  
Rna Sequence ◽  
E Coli ◽  
A Tec ◽  
And Function

RNA folds into secondary and tertiary structures that can mediate diverse cellular functions. Understanding how RNA sequence directs the formation of biologically active structures requires approaches that can comprehensively assess how changes in an RNA sequence affect its structure and function. Towards this goal, I have developed a general method for purifying E. coli RNA polymerase (RNAP) transcription elongation complexes (TECs) for use in systematic RNA assays. My approach depends on two constituent technologies: First, I have designed an E. coli σ70 promoter that can be efficiently barcoded using a one-pot series of enzymatic reactions. Second, I have developed a strategy for purifying promoter-initiated E. coli RNAP TECs by selective photo-elution from streptavidin-coated magnetic beads. Together, these methods establish a platform for the development of TEC Display assays in which the functional properties of RNA sequence variants can be recorded by fractionating and quantitatively barcoding a TEC library.

scholarly journals Purification of synchronized E. coli transcription elongation complexes by reversible immobilization on magnetic beads

2022 ◽  
Author(s):  
Skyler L. Kelly ◽  
Courtney E. Szyjka ◽  
Eric J. Strobel
Keyword(s):  
Rna Polymerase ◽  
High Performance ◽  
Magnetic Beads ◽  
Solid Phase ◽  
Transcription Elongation ◽  
E Coli ◽  
First Occurrence ◽  
Transcription Antitermination ◽  
Dna Strand

Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more NTPs from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes, but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 5′ leader sequence, called C3-SC1, to reversibly capture synchronized TECs on magnetic beads. We first show that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating NTPs), and compatible with solid-phase transcription; the yield of this purification is ~8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP-sensing and ppGpp-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

scholarly journals Structure and Function of the Transcription Elongation Factor GreB Bound to Bacterial RNA Polymerase

Cell ◽  
2003 ◽  
Vol 114 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Natacha Opalka ◽  
Mark Chlenov ◽  
Pablo Chacon ◽  
William J. Rice ◽  
Willy Wriggers ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structure And Function ◽  
Transcription Elongation Factor ◽  
Bacterial Rna ◽  
And Function

scholarly journals Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides

1994 ◽  
Vol 303 (2) ◽  
pp. 407-411 ◽  
Author(s):  
J F Van Iwaarden ◽  
J C Pikaar ◽  
J Storm ◽  
E Brouwer ◽  
J Verhoef ◽  
...  
Keyword(s):  
Lipid A ◽  
Magnetic Beads ◽  
Protein A ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Carbohydrate Binding ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

scholarly journals Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation

2015 ◽  
Vol 59 (10) ◽  
pp. 6195-6202 ◽  
Author(s):  
Jenny Yang ◽  
Thirunavukkarasu Annamalai ◽  
Bokun Cheng ◽  
Srikanth Banda ◽  
Rakhi Tyagi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Topoisomerase I ◽  
Mutation Frequency ◽  
Antimicrobial Agents ◽  
Transcription Elongation ◽  
Terminal Point ◽  
Content Type ◽  
E Coli ◽  
Network Response

ABSTRACTTopoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) ofEscherichia colitopoisomerase I interact directly with RNA polymerase to remove transcription-driven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study,Escherichia coliwith atopA66single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by thistopA66mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in thetopA66mutant. The increased antibiotic sensitivity for thetopA66mutant can be reversed by the expression of recombinantE. colitopoisomerase I but not by the expression of recombinantMycobacterium tuberculosistopoisomerase I that has a nonhomologous CTD even though the recombinantM. tuberculosistopoisomerase I can restore most of the plasmid DNA linking number deficiency caused by thetopA66mutation. Direct interactions ofE. colitopoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response.

scholarly journals NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Matti Turtola ◽  
Georgiy A Belogurov
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Transcription Elongation ◽  
Single Nucleotide ◽  
E Coli ◽  
Transcription Bubble ◽  
Nucleotide Addition ◽  
Nascent Rna ◽  
Comprehensive Mapping

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.

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