Structure of the LexA repressor-DNA complex probed by affinity cleavage and affinity photo-cross-linking

Biochemistry
P DumoulinM Schnarr

Abstract

The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage. These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator. In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein. In all four cases, the original amino acid was an arginine. The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as inferred from the structure of the LexA DNA binding domain [Fogh et al. (1994) EMBO J. 13, 3936-3944]. In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA. The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid. To reduce hydroxyl radical diffusion, the EDTA-iron cleavage reactions were done in the presence of high amounts of glycerol. The resul...Continue Reading

References

Nov 20, 1992·Journal of Molecular Biology·K L RolandJ W Little
Nov 1, 1991·Journal of Bacteriology·A Raymond-Denise, N Guillen
Aug 1, 1990·Molecular & General Genetics : MGG·P Oertel-BuchheitM Granger-Schnarr
Jul 1, 1985·Journal of Bacteriology·K F Wertman, D W Mount
Mar 20, 1993·Journal of Molecular Biology·Y Chen, R H Ebright

❮ Previous
Next ❯

Citations

Nov 5, 2005·Nucleic Acids Research·Eli S GrobanCharles M Lovett
Sep 19, 2002·Protein Science : a Publication of the Protein Society·Gaoping XiaoChi-Hong B Chen
Jan 19, 2005·The FEBS Journal·Aalt D J van DijkAlexandre M J J Bonvin
Mar 4, 1997·Proceedings of the National Academy of Sciences of the United States of America·K MurakamiA Ishihama
Sep 30, 2004·Biophysical Journal·F KühnerH E Gaub
Apr 26, 2000·Chemistry & Biology·D A Erlanson
Oct 19, 2006·The Journal of Biological Chemistry·Mariam QuinonesMatthew K Waldor
Jan 27, 2010·Chemical Reviews·Marguerite Pitié, Geneviève Pratviel

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.