Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti - A combined molecular biology and force spectroscopy investigation

Frank Wilco Bartels, Birgit Baumgarth, Dario Anselmetti, Robert Ros, Anke Becker

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of koff=(1.2±1.0)×10-3s-1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.

Original languageEnglish (US)
Pages (from-to)145-152
Number of pages8
JournalJournal of Structural Biology
Volume143
Issue number2
DOIs
StatePublished - Aug 1 2003
Externally publishedYes

Fingerprint

Sinorhizobium meliloti
Multigene Family
Genetic Promoter Regions
Molecular Biology
Spectrum Analysis
Carrier Proteins
Symbiosis
Medicago sativa
DNA-Binding Proteins
Gram-Negative Bacteria
Proteins
Nitrogen
Soil
Hot Temperature
Gene Expression
DNA
Genes
galactoglucan
Single Molecule Imaging

Keywords

  • Atomic force microscopy
  • Galactoglucan
  • Protein-DNA interaction
  • Regulatory protein
  • Single molecule force spectroscopy
  • Sinorhizobium meliloti

ASJC Scopus subject areas

  • Structural Biology

Cite this

Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti - A combined molecular biology and force spectroscopy investigation. / Bartels, Frank Wilco; Baumgarth, Birgit; Anselmetti, Dario; Ros, Robert; Becker, Anke.

In: Journal of Structural Biology, Vol. 143, No. 2, 01.08.2003, p. 145-152.

Research output: Contribution to journalArticle

@article{3bd5cc5762fa497f9da24cdb809e6401,
title = "Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti - A combined molecular biology and force spectroscopy investigation",
abstract = "Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of koff=(1.2±1.0)×10-3s-1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.",
keywords = "Atomic force microscopy, Galactoglucan, Protein-DNA interaction, Regulatory protein, Single molecule force spectroscopy, Sinorhizobium meliloti",
author = "Bartels, {Frank Wilco} and Birgit Baumgarth and Dario Anselmetti and Robert Ros and Anke Becker",
year = "2003",
month = "8",
day = "1",
doi = "10.1016/S1047-8477(03)00127-8",
language = "English (US)",
volume = "143",
pages = "145--152",
journal = "Journal of Structural Biology",
issn = "1047-8477",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti - A combined molecular biology and force spectroscopy investigation

AU - Bartels, Frank Wilco

AU - Baumgarth, Birgit

AU - Anselmetti, Dario

AU - Ros, Robert

AU - Becker, Anke

PY - 2003/8/1

Y1 - 2003/8/1

N2 - Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of koff=(1.2±1.0)×10-3s-1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.

AB - Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of koff=(1.2±1.0)×10-3s-1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.

KW - Atomic force microscopy

KW - Galactoglucan

KW - Protein-DNA interaction

KW - Regulatory protein

KW - Single molecule force spectroscopy

KW - Sinorhizobium meliloti

UR - http://www.scopus.com/inward/record.url?scp=0042335817&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0042335817&partnerID=8YFLogxK

U2 - 10.1016/S1047-8477(03)00127-8

DO - 10.1016/S1047-8477(03)00127-8

M3 - Article

C2 - 12972351

AN - SCOPUS:0042335817

VL - 143

SP - 145

EP - 152

JO - Journal of Structural Biology

JF - Journal of Structural Biology

SN - 1047-8477

IS - 2

ER -