Transcriptional Regulation by Antitermination. Interaction of RNA with NusB Protein and NusB/NusE Protein Complex of Escherichia coli

Lüttgen, Holger; Robelek, Rudolf; Mühlberger, R.; Diercks, Tammo; Schuster, Stephan C.; Kohler, Peter O.; Kessler, Horst; Bacher, Adelbert; Richter, Gerald

doi:10.1006/jmbi.2001.5388

Cited by 34 publications

(29 citation statements)

References 29 publications

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“…2D). This value was also confirmed by MALDI-TOF mass spectrometry (data not shown) and is consistent with previous studies and with a monomer of 15.8 kDa based on the amino acid residue sequence (24,32).…”

Section: Resultssupporting

confidence: 92%

“…coli NusE was purified as described above. Despite previous reports that this protein is insoluble in its native form (24,33), we note that concentrations of NusE up to a concentration of ϳ20 M could be obtained by slow dialysis through a urea gradient (from 6 M to native buffer). Previous data from proteolytic digests and chemical degradation experiments had suggested that free E. coli NusE might exist in a relatively unstructured form (34), and because NusE from Mycobacterium tuberculosis has also been shown (by CD spectroscopy and NMR) to be relatively unstructured (even when bound to NusB (35)), it seems likely that E. coli NusE is also largely unstructured.…”

Section: Resultscontrasting

confidence: 74%

“…Converting the c(s) profile into c(M) data revealed an estimated molecular mass of ϳ27 kDa for the 2.2 S species (Fig. 4B), suggesting that the NusB and NusE proteins do interact to form a heterodimer with a stoichiometry of 1:1 (24).…”

Section: Resultsmentioning

confidence: 99%

“…Binding of NusB and NusE (S10) to boxA RNA complex that is involved in the N protein-dependent antitermination events that regulate the lytic growth of lambdoid phages. Rigorous thermodynamic binding assays using fluorescence anisotropy and analytical ultracentrifugation involve fewer assumptions and have greater sensitivity than the gel shift and surface plasmon resonance assays used to measure binding in previous studies (4,24), and have permitted us to analyze the assembly process as a total system of three separate components involving several constituent binary interactions. In addition, the fact that we have been able to obtain and examine soluble free E. coli NusE, although over a limited concentration range, has been crucial to obtaining estimates of binding parameters for the various equilibria listed in Equations 2-7.…”

Section: Discussionmentioning

confidence: 99%

“…Earlier studies (4,23,24) of this assembly reaction, carried out using gel shift and surface plasmon resonance assays, have been interpreted in terms of a sequential two-step assembly mechanism for the formation of the NusB-NusE-boxA RNA complex (Fig. 1A).…”

mentioning

confidence: 99%

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Assembly of an RNA-Protein Complex

Greive

Lins²,

Hippel

2005

Journal of Biological Chemistry

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Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K d values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K d values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available singlestranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.Macromolecular machines involving multiple protein and nucleic acid components drive and regulate many important cellular processes. These include gene expression, protein synthesis and trafficking, as well as signal transduction and many other processes. The assembly of the relevant macromolecular complexes is often controlled by additional regulatory factors that modulate the effective binding affinity of the central components of a self-assembling macromolecular machine. Another regulatory strategy involves controlling the local concentration of a critical component of the complex, which can perturb crucial binding interactions by manipulating mass action effects. In principle this strategy uses changes in the concentration parameter as a "switch" to turn the targeted assembly reaction on or off; a good example of this strategy involves using the control of the synthesis (and thus of the free concentration) of the single-stranded DNA-binding protein of T4 (gp32) to regulate the assembly of the functional T4 DNA replication complex (1). Furthermore, if this component is "tethered" in the vicinity of the assembly to be regulated (e.g. by cis-RNA looping (2, 3)), this switch can be used to confine the regulation of concentration change only to nearby target loci on the genome and not to others.Characterizing the binding interactions between particular components of a macromolecular complex and elucidating the mechanisms of assembly is a challenging, but vital, part of understanding how cellular processes are regulated. This problem is compounded when...

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Section: Resultssupporting

confidence: 92%

Section: Resultscontrasting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Assembly of an RNA-Protein Complex

Greive

Lins²,

Hippel

2005

Journal of Biological Chemistry

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NMR Chemical Shift Perturbation Study of the N‐Terminal Domain of Hsp90 upon Binding of ADP, AMP‐PNP, Geldanamycin, and Radicicol

et al. 2003

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Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.

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A survey of the year 2002 commercial optical biosensor literature

Rich

Myszka

2003

J of Molecular Recognition

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We have compiled 819 articles published in the year 2002 that involved commercial optical biosensor technology. The literature demonstrates that the technology's application continues to increase as biosensors are contributing to diverse scientific fields and are used to examine interactions ranging in size from small molecules to whole cells. Also, the variety of available commercial biosensor platforms is increasing and the expertise of users is improving. In this review, we use the literature to focus on the basic types of biosensor experiments, including kinetics, equilibrium analysis, solution competition, active concentration determination and screening. In addition, using examples of particularly well-performed analyses, we illustrate the high information content available in the primary response data and emphasize the impact of including figures in publications to support the results of biosensor analyses.

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Transcriptional Regulation by Antitermination. Interaction of RNA with NusB Protein and NusB/NusE Protein Complex of Escherichia coli

Cited by 34 publications

References 29 publications

Assembly of an RNA-Protein Complex

Assembly of an RNA-Protein Complex

NMR Chemical Shift Perturbation Study of the N‐Terminal Domain of Hsp90 upon Binding of ADP, AMP‐PNP, Geldanamycin, and Radicicol

A survey of the year 2002 commercial optical biosensor literature

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