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M Buck

Publications and source records attributed to M Buck.

At least 55 records · Page 3Linked to original sources
PubMed2000

Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus.

The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) and the NifA homologue, AQ_218. Purified A. aeolicus sigma(N) bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFDeltaHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.

Amino Acid Sequence↗
PubMed2000

c-Myb modulates transcription of the alpha-smooth muscle actin gene in activated hepatic stellate cells.

Expression of alpha-smooth muscle actin (alpha-SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of alpha-SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal alpha-SMA E box. Therefore, we analyzed the role of c-Myb in alpha-SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the -724 and -271 alpha-SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (-223/-216 bp). Alpha-SMA/LUC minigenes with mutation (-219/-217 bp), truncation (-224 bp), or deletion (-191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a approximately 10-fold stimulation of -724 alpha-SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys(43) --> Asp) or a c-Myb antisense RNA blocked transcription from the -724 alpha-SMA/LUC or -271 alpha-SMA/LUC in activated cells. Moreover, transfection of c-myb antisense, but not sense, RNA inhibited both expression of the endogenous alpha-SMA gene and stellate cell activation, whereas transfection of c-myb stimulated alpha-SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys(43) in c-Myb is required for this effect.

Actins↗
PubMed1999

The C-terminal 12 amino acids of sigma(N) are required for structure and function.

The sigma(N) protein is an alternative sigma subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid "tail" at the C-terminus of Klebsiella pneumoniae sigma(N), which was predicted to be largely surface-exposed and to be mostly loop (that is not alpha-helical or beta-strand). Deletion of this tail from N-terminal hexahistidine-tagged sigma(N) led to loss of sigma(N)-dependent transcription activity in vivo. We overexpressed and purified this deletion-mutant protein for in vitro characterization. The purified deleted protein showed decreased RNA polymerase core- and DNA-binding activities compared to the full-length protein and transcription activity was greatly impaired. Furthermore, evidence from circular dichroism and protease digestion experiments together suggested that deletion of the C-terminus tail resulted in a loss of conformational constraint in the protein. We discuss a possible structural role for the 12 amino acids at the C-terminus of sigma(N).

Amino Acid Sequence↗
PubMed1999

Systematic analysis of sigma54 N-terminal sequences identifies regions involved in positive and negative regulation of transcription.

The conserved amino-terminal region of sigma 54 (Region I) contains sequences that allow response to activator proteins, and inhibit initiation in the absence of activator. Alanine-scanning mutagenesis has been used to systematically define Region I elements that contribute to each of these functions. Amino acid residues from 6 to 50 were substituted with alanine in groups of three consecutive residues, making a total of 15 mutants. Mutants were tested for their ability to mediate activation in vivo, and in vitro, and to support transcription in the absence of activator in vitro. Most mutations located between residues 15 and 47 altered sigma function, while mutations between residues 6 and 14, and 48-50 had little effect. The defective mutants ala 15-17, 42-44, and 45-47 define new amino acids required for normal sigma function. In general, there is an inverse correlation between the levels of activated and activator-independent transcription, suggesting that the two functions are linked. When activated, the defective sigma mutants, except for ala 24-26, formed heparin-resistant open complexes similar to wild-type sigma. Mutant ala 24-26 formed heparin-unstable open complexes, suggesting that this mutation interferes with a different step in the initiation pathway.

Amino Acid Sequence↗
PubMed1999

Functions of the sigma(54) region I in trans and implications for transcription activation.

Control of transcription frequently involves the direct interaction of activators with RNA polymerase. In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation. Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme (and not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization. Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54). Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

Bacterial Proteins↗
PubMed1999

Characterisation of holoenzyme lacking sigmaN regions I and II.

The sigma-N (sigmaN) protein associates with bacterial core RNA polymerase to form a holoenzyme that is silent for transcription in the absence of enhancer-binding activator proteins. Here we show that the acidic Region II of sigmaN from Klebsiella pneumoniae is dispensable for polymerase isomerisation and trans-cription under conditions where the inhibited state of the holoenzyme is relieved by removal of sigmaN Region I sequences. Holoenzymes lacking Region I or Regions I+II were equally susceptible to the order of addition-dependent inhibition or stabilisation of DNA binding afforded by in trans Region I sequences. Region I+II-deleted [sigma] formed a holoenzyme with a DNA-binding activity more susceptible to inhibition by non-specific DNA than that lacking Region I. Region II sequences appear more closely associated with formation of a holoenzyme and [sigma] proficient in DNA binding than with changes in holoenzyme conformation needed for unmasking a single-strand DNA-binding activity used for open complex for-mation. Region II may therefore function to optimise DNA interactions for an efficient sigma cycle.

Base Sequence↗
PubMed1999

Sequences in sigmaN determining holoenzyme formation and properties.

Sigma subunits of bacterial RNA polymerases are closely involved in many steps of promoter-specific transcription initiation. Holoenzyme formed with the specialised minor sigma-N (sigmaN) protein binds rare promoters in a transcriptionally inactive state and functions in enhancer-dependent transcription. Using competition and dissociation assays, we show that sigmaN-holoenzyme has a stability comparable to the major sigma70-holoenzyme. Purified partial sequences of sigmaN were prepared and assayed for retention of core RNA polymerase binding activity. Two discrete fragments of sigmaN which both bind the core but with significantly different affinities were identified, demonstrating that the sigmaN interface with core RNA polymerase is extensive. The low affinity segment of sigmaN included region I sequences, an amino terminal domain which mediates activator responsiveness and formation of open promoter complexes. The higher affinity site lies within a 95 residue fragment of region III. We propose that the core to region I contact mediates properties of the sigmaN-holoenzyme important for enhancer responsiveness. Heparin is shown to dissociate sigmaN and core, indicating that disruption of the holoenzyme is involved in the heparin sensitivity of the sigmaN closed complex.

Amino Acid Sequence↗
PubMed1999

Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization.

In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

DNA↗
PubMed1999

Secondary structure of the C-terminal DNA-binding domain of the transcriptional activator NifA from Klebsiella pneumoniae: spectroscopic analyses.

The conformation of the C-terminal DNA-binding domain of the transcriptional activator NifA from Klebsiella pneumoniae has been probed by circular dichroism (CD), Fourier-transformed infrared (FT-IR), and nuclear magnetic resonance (NMR) spectroscopy in combination. Secondary structure prediction suggests that the C-terminal half of the domain contains three alpha-helices. The spectra show that the domain is folded in the absence of DNA and of the N-terminal and central domains of NifA. The three spectroscopic techniques suggest slightly different proportions of secondary structural elements but all suggest that it contains about 33% alpha-helix. These results are in agreement with a previous prediction suggesting that NifA contains a helix-turn-helix motif and with the amount of alpha-helix predicted. The environment of the aromatic residues was examined by CD and NMR spectroscopy, which suggest that one or both of the tryptophan residues are involved in the tertiary structure of the protein but that the tyrosine residue in the helix-turn-helix motif is solvent exposed and so available to bind to DNA. The thermal melting profiles and pH-dependent structural changes were also examined by CD spectroscopy. This technique indicates that at low pH there is an increase in the secondary structure and interactions contributing to the tertiary structure. Many of the acidic residues are predicted to be on a single helix, before the helix-turn-helix motif, which may therefore be important for maintaining the structure and function of the C-terminal peptide; alternatively, the N-terminal half of the domain may become more folded at low pH.

Amino Acid Sequence↗
PubMed1999

Region I modifies DNA-binding domain conformation of sigma 54 within the holoenzyme.

Activation of transcription at sigma 54-dependent bacterial promoters proceeds via a mechanism that is independent of recruitment of RNA polymerase to the promoter, but is instead totally dependent on activator-driven conformational changes in the promoter-bound RNA polymerase. Understanding of the activation mechanism first requires a detailed description of the interactions taking place in the polymerase holoenzyme and closed complex. The interactions of sigma 54 with core RNA polymerase and promoter DNA were investigated using enzymatic and chemical (hydroxyl radical) protease footprinting of sigma. Regions of sigma were identified that are in direct contact with ligands, or whose conformation changes following ligand binding. A comparison of wild-type sigma and a mutant bearing a deletion of conserved Region I, which is required for response to activator proteins and regulated initiation, revealed differences in the protease sensitivity of free sigma indicating that Region I affects sigma conformation. Comparison of the holoenzyme and closed complex hydroxyl radical footprints revealed that residues of wild-type sigma protected by promoter DNA overlap, to a large extent, the residues of Region I-deleted sigma protected by core polymerase. Region I could thus modify DNA-binding by changing conformation of the DNA-binding domain of sigma 54 in a core polymerase-dependent manner. These differences can account for the modified promoter binding of the Region I-deleted sigma holoenzyme observed by DNA footprinting, and are likely of significance to the Region I-dependent activation of transcription.

Binding Sites↗
PubMed1999

Phosphorylation of rat serine 105 or mouse threonine 217 in C/EBP beta is required for hepatocyte proliferation induced by TGF alpha.

We report that TGF alpha induces activation of the p90 ribosomal S kinase (RSK), which results in the phosphorylation of rat C/EBP beta on Ser-105 and of mouse C/EBP beta on Thr-217 and concomitantly stimulates proliferation in differentiated hepatocytes. Moreover, C/EBP beta-/- mouse hepatocytes respond to TGF alpha when wild-type C/EBP beta is reexpressed, whereas they remain refractory to the growth effect of TGF alpha when expressing phosphoacceptor mutants rat C/EBP beta Ala-105 or mouse C/EBP beta Ala-217. In contrast, C/EBP beta-/- hepatocytes expressing the phosphorylation mimic mutants, rat C/EBP beta Asp-105 or mouse C/EBP beta Glu-217, exhibited marked proliferation in the absence of TGF alpha. Thus, a site-specific phosphorylation of the transcription factor C/EBP beta is critical for hepatocyte proliferation induced by TGF alpha and other stimuli that activate RSK.

Amino Acid Sequence↗
PubMed1999

Involvement of the sigmaN DNA-binding domain in open complex formation.

sigmaN (sigma54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae sigmaN mutants, altered in the carboxy DNA-binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent transcription. The mutant holoenzymes have altered activator-dependent interactions with promoter sequences that normally become melted. Activator-dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I sigmaN, suggesting different defects. The DNA-binding domain substitutions are within sigmaN sequences previously shown to be buried upon formation of the wild-type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the sigmaN-core and sigmaN-DNA interfaces has occurred to change holoenzyme activity. Core-binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of sigmaN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator-dependent transcription, formed a stable activator-independent holoenzyme promoter complex on premelted DNA templates.

Bacterial Proteins↗
PubMed1999

The sigma 54 DNA-binding domain includes a determinant of enhancer responsiveness.

The bacterial sigma54 protein associates with core RNA polymerase to form a holoenzyme that functions in enhancer-dependent transcription. Isomerization of the sigma54 polymerase and its engagement with melted DNA in open promoter complexes requires nucleotide hydrolysis by an enhancer-binding activator. We show that a single amino acid substitution, RA336, in the Klebsiella pneumoniae sigma54 C-terminal DNA-binding domain allows the holoenzyme to isomerize, engage with stably melted DNA and to transcribe from transiently melting DNA without an activator. Activator responsiveness for the formation of stable open complexes remained intact. The activator-independent transcription phenotype of RA336 is shared with mutants in amino-terminal Region I sequences. Thus, in sigma54, two distinct domains function for enhancer responsiveness. A sigma54-DNA contact mediated by R336 appears to be part of a network of interactions necessary for maintaining the transcriptionally inactive state of the holoenzyme. We suggest activator functions to change these interactions and facilitate open complex formation through promoting polymerase isomerization.

Amino Acid Substitution↗
PubMed1999

A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle.

In this study replication of A-type and B-type Epstein-Barr virus (EBV) strains has been assessed. A-type and B-type type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting. All of the B-type EBV strains were capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast the A-type strains, other than strain IARC-BL36, did not readily produce VCA in any of the different donor lymphocytes used. This study demonstrates another biological difference between the two virus types: their ability to spontaneously enter the lytic cycle.

Animals↗
PubMed1999

Epstein-Barr virus-encoded RK-BARF0 protein expression.

The cellular localization of the Epstein-Barr virus-encoded RK-BARF0 protein was analyzed by fluorescence microscopy and immunoblotting. The recombinant RK-BARF0 protein was found to be tightly bound to nuclear structures, whereas 16- to 20-kDa RK-BARF0 derivatives, generated by differential splicing of the RK-BARF0 transcript, were present throughout the cell. Moreover, a previously generated anti-RK-BARF0 rabbit serum was found to cross-react with cellular proteins, showing that the previously identified 30- to 35-kDa membrane-associated proteins do not represent RK-BARF0.

Animals↗
PubMed1999

Intron retention may regulate expression of Epstein-Barr virus nuclear antigen 3 family genes.

The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4, and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.

Alternative Splicing↗