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Biological NMR Spectroscopy$
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John L. Markley and Stanley J. Opella

Print publication date: 1997

Print ISBN-13: 9780195094688

Published to Oxford Scholarship Online: November 2020

DOI: 10.1093/oso/9780195094688.001.0001

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Heteronuclear Strategies for the Assignment of Larger protein/DNA complexes: Application to the 37 kDa trp Represser-Operator Complex

Heteronuclear Strategies for the Assignment of Larger protein/DNA complexes: Application to the 37 kDa trp Represser-Operator Complex

Chapter:
(p.50) 6 Heteronuclear Strategies for the Assignment of Larger protein/DNA complexes: Application to the 37 kDa trp Represser-Operator Complex
Source:
Biological NMR Spectroscopy
Author(s):

M.J. Revington

W. Lee

Publisher:
Oxford University Press
DOI:10.1093/oso/9780195094688.003.0012

The sequence-specific DNA binding function of many proteins is recognized as one of the central mechanisms of regulating transcription and DNA replication and repair. The ability of these proteins to select a short (usually 10 to 20 basepair) sequence out of the entire genome with which to form a stable complex is a prime example of molecular recognition. Atomic resolution structural studies using NMR and X-ray crystallography have emerged as essential techniques in understanding the basis of specificity and stability in these systems. While NMR studies of small DNA-binding domains of proteins have become almost routine (see Kaptein, 1993 for a review) relatively few NMR studies of protein-DNA complexes have been reported. These include the lac repressor headpiece complex (Chuprina et al., 1993). the Antennapedia homeodomain complex (Billetere et al., 1993), the GATA-1 complex (Omichinski et al., 1993). and the Myb DNA binding domain complex (Ogata et al., 1993); all of these complexes are smaller than 20 kDa. In most cases, size limitations have meant that only the DNA binding domain of the protein in complex with a single binding element have been studied. In vivo, however, most DNA binding proteins are much larger than these domains and often function as oligomers. The decrease in quality and increase in complexity of spectra as the molecular weight of the sample increases, limits the number of systems amenable to study using NMR and influences the decision to focus on single domains of multidomain proteins. However, since many DNA-binding proteins are regulated by the binding of ligands, other proteins or phosphorylation, often at sites distal from the DNA-binding domain, it is preferable to study as much of the intact protein as possible in order to characterize allosteric and regulatory mechanisms (Pabo and Sauer, 1992). E. coli trp repressor is a 25 kDa homodimer that regulates operons involved in tryptophan biosynthesis. The dimer is one of the smallest intact proteins that binds sequence specifically to DNA and whose affinity is modulated by an effector (L-tryptophan).

Keywords:   operator DNA, trp repressor

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