lac represser binding to non-operator DNA: Detailed studies and a comparison of equilibrium and rate competition methods

Lin, Syr-Yaung; Riggs, Arthur D.

doi:10.1016/0022-2836(72)90184-2

Cited by 313 publications

(176 citation statements)

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“…Phosphorus imaging analysis was used to quantitate the bands corresponding to bound and unbound probe. The competitive binding of a protein to the labeled RNA probe and the unlabeled competitor RNA can be described by Equation 2 (33), where K rel is the ratio of K t , the apparent dissociation constant of the labeled probe, to K C , the apparent dissociation constant of the competitor, and P t , T t , and C t are the concentrations of the protein, the radiolabeled probe, and the competitor probe, respectively. Competition data were fit to Equation 2 by using KaleidaGraph 3.5 software.…”

Section: Methodsmentioning

confidence: 99%

Contributions of the Individual Domains in Human La Protein to Its RNA 3′-End Binding Activity

Ohndorf

Steegborn

Knijff

et al. 2001

Journal of Biological Chemistry

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The autoantigen La regulates the maturation of RNA polymerase III transcripts by binding to their poly(U) termination signal. The modular protein harbors a Nterminal RNA recognition motif (RRM), RRM1, and in the C-terminal domain, a second, atypical RRM2, in addition to a phosphorylation site, and a putative nucleotide binding site. This study presents a detailed investigation into the RNA 3-end binding properties of La by using binding titration and competition assays with subsequent gel mobility shift analysis. Two truncation mutants containing one (La-RRM1) or both (La-RRM1-RRM2) RNA-binding domains were constructed, overexpressed, and purified. A K d value of 25 ؎ 10 nM for La binding to a nonameric RNA ligand with the oligouridylate recognition sequence was obtained, discriminating with a specificity ratio of ϳ100 for this probe over a RNA ligand with a 3-poly(A) stretch. The N-terminal La-RRM1 region was identified as the major contributor of these properties to La, manifested in a 5-fold lower K d of 5 ؎ 3 nM and a slightly increased specificity ratio of 120 for the RNA ligand. The atypical RRM2 in the C-terminal domain of La has an unprecedented negative effect on 3-end RNA recognition, as indicated by a higher K d value of 90 ؎ 10 nM for the La-RRM1-RRM2 mutant but comparable specificity. Thus the C-terminal regions beyond RRM2 positively modulate the RNA binding affinity of La. Negative regulation, however, occurs through Ser 366 phosphorylation decreasing the binding affinity by 2-fold. ATP had no influence on RNA complex formation. The functional implications of these findings for the mechanism of action of La are discussed.Human La protein (lupus antigen or La/SS-B) was originally identified as a major target of the autoimmune response in patients suffering from the autoimmune diseases Sjögren's syndrome and systemic lupus erythematosus (1). It is a conserved RNA-binding protein that recognizes specifically 3Ј-oligouridylate stretches (2). Associated with all RNA polymerase III transcripts where the 3Ј-UUU-OH sequence is added as the transcription termination signal, La is thought to play a central role in the metabolism of these RNAs by acting as a molecular chaperone that stabilizes and/or structures them for further processing (3). In HeLa cell extracts, the 5Ј-and 3Ј-end processing of tRNA precursors is influenced by La (4), and in vitro, La activity has been suggested in RNA polymerase termination, recycling, and initiation reactions (5-8). Furthermore, a Walker A nucleotide-binding motif in La was postulated to confer ATP-dependent RNA/DNA and RNA/RNA helicase activity (9, 10), but recently, the binding of La to double-stranded nucleic acids was shown to be independent of this site, and La does not appear to unwind double-stranded RNA (11). The Walker A motif has instead been implicated in binding to the 5Ј-triphosphate ends of nascent tRNAs, with the affinity negatively modulated by phosphorylation of Ser 366 (4, 7). Cytoplasmic functions for La have also been described, including facilitatio...

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Section: Methodsmentioning

confidence: 99%

Contributions of the Individual Domains in Human La Protein to Its RNA 3′-End Binding Activity

Ohndorf

Steegborn

Knijff

et al. 2001

Journal of Biological Chemistry

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“…If the competitor competes for binding to Vps45p, the fraction of bound Tlg2p(37-318)* decreases as the concentration of competitor increases. The apparent affinity (K c,app ) of the Vps45p-competitor interaction is calculated from a fit of the data to the Lin and Riggs equation (28).…”

Section: Vps45pmentioning

confidence: 99%

The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p

Furgason

MacDonald

Shanks

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The equilibrium binding constant was measured using the competition method (25,26). The wild-type DNA (2 pmol) was labeled (see above) and radioactive ATP was removed by passing the reaction mixture through a Sephadex G-50 column equilibrated with 10mM Tris, pH 7.6,150mM KCl andO.1 mM EDTA.…”

Section: Competition Equilibrium Dissociation Assaysmentioning

confidence: 99%

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Zhang

Gottlieb

1995

Nucl Acids Res

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Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCI, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 250C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCI to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Iobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution op the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system.

show abstract

lac represser binding to non-operator DNA: Detailed studies and a comparison of equilibrium and rate competition methods

Cited by 313 publications

References 10 publications

Contributions of the Individual Domains in Human La Protein to Its RNA 3′-End Binding Activity

Contributions of the Individual Domains in Human La Protein to Its RNA 3′-End Binding Activity

The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

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