Solution Structure of the HU Protein fromBacillus stearothermophilus

Vis, Hans; Mariani, Matteo; Vorgias, Constantin E.; Wilson, K.S.; Kaptein, Robert; Boelens, Rolf

doi:10.1006/jmbi.1995.0648

Cited by 103 publications

(82 citation statements)

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“…The precise details of this turn were not apparent in the original 3 A Ê structure, where it was thought to be more extensive (residues 14±20). It is now clear that residues 14±15 and 17±20 form the termini of the¯anking -helices, which agrees with more recent NMR results on HU (Vis et al, 1995).…”

Section: Monomer±monomer Interactionssupporting

confidence: 90%

“…This suggestion is supported by the fact that alanines and prolines dominate the hinge/bend region in all known HU sequences. An NMR analysis of HU has revealed the arm conformation, which agrees well with the region which is visible in our X-ray structure (Vis et al, 1995).…”

Section: The Saddle and The Armssupporting

confidence: 87%

“…We had originally predicted that the arm would form a simple -ribbon and that Pro63 would occupy position 2 of a -turn at the end. Recent NMR results on HU (Vis et al, 1995) have shown that our basic idea of a -ribbon is correct, but the details of the arm structure are not. Pro63 is now known to occupy position 1 of a type I -turn.…”

Section: Monomer±monomer Interactionsmentioning

confidence: 96%

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The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus

White

Wilson

Appelt

et al. 1999

Acta Crystallogr D Biol Cryst

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Protein HU is a ubiquitous prokaryotic protein which controls the architecture of genomic DNA. It binds DNA nonspeci®cally and promotes the bending and supercoiling of the double helical structure. HU is involved in many DNAassociated cellular processes, including replication, transcription and the packaging of DNA into chromosome-like structures. Originally determined at medium resolution, the crystal structure of HU has now been re®ned at 2.0 A Ê resolution. The high-resolution structure shows that the dimeric molecule is essentially a compact platform for twō exible and basic arms which wrap around the DNA molecule.To maximize the protein's stability, non-secondary structural regions are reduced to a minimum, there is an extensive aromatic hydrophobic core and several salt bridges and hydrogen-bonded water molecules knit together crucial regions. Based on the original medium-resolution structure of HU, several proposals were made concerning the structural basis of HU's ability to bind, bend and supercoil DNA. Each of these proposals is fully supported by the high-resolution structure. Most notably, the surfaces of the molecule which appear to mediate protein±DNA and protein±protein interactions have the ideal shapes and physicochemical properties to perform these functions.

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Section: Monomer±monomer Interactionssupporting

confidence: 90%

Section: The Saddle and The Armssupporting

confidence: 87%

Section: Monomer±monomer Interactionsmentioning

confidence: 96%

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The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus

White

Wilson

Appelt

et al. 1999

Acta Crystallogr D Biol Cryst

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“…1. A nearly complete backbone assignment (listed in Table I) was obtained using double and triple resonance methods, essentially in the manner described by Vis et al (23). Thus, amide 15 N and 1 H frequencies were obtained from a highresolution 600 MHz 1 H 15 N-HSQC spectrum, followed by the collection of corresponding intra-and inter-residual C ␣ and CO connectivities from 600 MHz HNCA, HN(CO)CA, HNCO, and HN(CA)CO spectra.…”

Section: Resultsmentioning

confidence: 99%

Identification of the Single-stranded DNA Binding Surface of the Transcriptional Coactivator PC4 by NMR

Werten¹,

Wechselberger²,

Boelens³

et al. 1999

Journal of Biological Chemistry

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The C-terminal domain of the eukaryotic transcriptional cofactor PC4 (PC4 CTD ) is known to bind with nanomolar affinity to single-stranded (ss)DNA. Here, NMR is used to study DNA binding by this domain in more detail. Amide resonance shifts that were observed in a 1 H 15 N-HSQC-monitored titration of 15 N-labeled protein with the oligonucleotide dT 18 indicate that binding of the nucleic acid occurs by means of two anti-parallel channels that were previously identified in the PC4 CTD crystal structure. The ␤-sheets and loops that make up these channels exhibit above average flexibility in the absence of ssDNA, which is reflected in higher values of T 1 , reduced heteronuclear nuclear Overhauser effects and faster deuterium exchange rates for the amides in this region. Upon ssDNA binding, this excess flexibility is significantly reduced. The binding of ssDNA by symmetry-related channels reported here provides a structural rationale for the preference of PC4 CTD for juxtaposed single-stranded regions (e.g. in heteroduplexes) observed in earlier work.The eukaryotic general transcriptional cofactor PC4 is known to enhance activated in vitro and in vivo transcription from various RNA polymerase II promoters, in concert with members of all major classes of transcriptional activators (1-3). The activator-dependent stimulation of transcription by PC4 has been shown to originate mostly from increased recruitment of basal transcription factors during the early stages of preinitiation complex (PIC) 1 formation (4). It is likely that this effect is caused by the interactions that have been reported between PC4 and the activation domains of promoter-binding activator proteins on the one hand, and basal factors (including TFIIA) on the other (1, 3). Thus, PC4 appears to act as a bridging factor that stabilizes the preinitiation complex.Recently it has become clear that PC4 has several additional functions. In the first place, the protein was found to be able to repress transcription under specific conditions (5-7). Repression at moderate PC4 concentrations is observed in minimal in vitro transcription systems, that normally do not require the basal factor TFIIH (5, 6), and with respect to aspecific transcription initiation by RNA polymerase II from DNA ends and unwound regions (6). Repression is not observed in full-factor in vitro transcription, because TFIIH is able to overcome repression by PC4 (5, 6). Thus, PC4 appears to act as what could be called a fidelity-enhancing factor for RNA polymerase II transcription initiation by repressing adventitious transcription from incomplete PICs on the one hand, and on the other hand enhancing transcription from fully assembled PICs, i.e. those that include TFIIH, generally believed to be the last factor to enter the PIC during assembly (8). Surprisingly, PC4 has recently also been found to associate in vivo with the RNA polymerase III transcription factor TFIIIC and to stimulate transcription by RNA polymerase III by increasing the rate of reinitiation (9). Interestingly, copurific...

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“…are homodimers of intertwined polypeptides containing 90 and 92 amino acid residues, respectively. Both the crystal and solution structures have been determined for HBst~White et al, 1989;Vis et al, 1995!. HBsu differs in sequence from HBst at only 12 of the amino acid positions, and by a two amino acid extension at the C-terminus. Nevertheless, the stabilities of the two proteins vary considerably under some conditions~Wilson et al, 1990;Kawamura et al, 1996!.…”

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confidence: 99%

Selective association of protein molecules followed by mass spectrometry

1999

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Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo-and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near-equilibrium conditions and will be relevant to a wide range of biological systems.

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Solution Structure of the HU Protein fromBacillus stearothermophilus

Cited by 103 publications

References 0 publications

The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus

The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus

Identification of the Single-stranded DNA Binding Surface of the Transcriptional Coactivator PC4 by NMR

Selective association of protein molecules followed by mass spectrometry

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