A simple procedure for visualising protein-nucleic acid complexes by photochemical crosslinking.
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Bacterial Proteins↗
Biomedical subjects
M Buck
Publications and source records attributed to M Buck.
LAP (NF-IL-6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation.
During postnatal liver development, LAP (NF-IL-6, C/EBP beta) expression and hepatocyte proliferation are mutually exclusive. In addition to transactivating liver-specific genes, LAP, but not C/EBP alpha, arrests the cell cycle before the G1/S boundary in hepatoma cells. LIP, a liver-inhibitory protein, which is translated from LAP mRNA lacking the activation domain of LAP, is not only ineffective in blocking hepatoma cell proliferation but also antagonizes the effect of LAP on the cell cycle. Deletion analysis indicated that this effect of LIP required only the DNA-binding and leucine zipper domains. In addition we found that integrity of the LAP dimerization and activation domains is indispensable for the arrest of cell proliferation induced by LAP. Thus, hepatocyte differentiation and its characteristic quiescent state may be modulated by the LAP/LIP ratio.
Amino Acid Sequence↗
Identification of a DNA-contacting surface in the transcription factor sigma-54.
The transcription factor sigma-54 (sigma 54) is a sequence-specific DNA-binding protein that directs RNA polymerase to a particular class of promoter. The interaction of sigma 54 with promoter DNA has been analysed by protein-DNA crosslinking and enzymatic and chemical proteolysis. Direct physical evidence for a DNA-contacting surface within the carboxy-terminal one-third of the protein has been obtained. This region of sigma 54 is likely to be close to the surface of the protein, and contacts DNA when either sigma 54 or the sigma 54-holoenzyme bind specifically to promoter DNA. The amino-terminal region of sigma 54 appears to be highly susceptible to proteolysis, and its integrity influences the accessibility towards proteolysis of a second region of sigma 54, which includes the DNA-contacting surface. Thus the amino-terminal region of sigma 54 may have a role in influencing its DNA-binding properties, the major determinants of which appear to reside in the carboxy-terminal one-third of the protein.
Bacterial Proteins↗
Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.
The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA.
Azotobacter vinelandii↗
LAP (NF-IL6) transactivates the collagen alpha 1(I) gene from a 5' regulatory region.
Although collagen is known to enhance hepatocyte differentiation and hepatocytes produce collagen in vivo, the transcriptional factors responsible for collagen type I gene expression in hepatic cells are not known. LAP (Liver Activator Protein) is a member of the C/EBP family, which in differentiated hepatocytes contributes to the high levels of liver-specific gene expression. In this study we show that LAP binds to the collagen alpha 1(I) promoter at both reverse CCAAT motifs and activates transcription. Furthermore, an upstream element, collagen element I (-370/-344), which shares homology with the LAP binding cis-element of the albumin promoter (9 of 13 bp) is described. This collagen element I stimulates transcription in both orientations and when placed in front of either a homologous or a heterologous chimeric report construct. These experiments suggest that LAP may be important in the expression of collagen in differentiated hepatocytes through both the promoter and a newly described upstream element.
Base Sequence↗
A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding.
The effect of 2,2,2-trifluoroethanol (TFE) on the solution conformation of hen egg white lysozyme has been investigated using circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. Addition of TFE to lysozyme at pH 2.0, 27 degrees C, up to a concentration of 15% (v/v) induces only slight changes in the NMR spectrum. However, above this concentration a cooperative transition to a new but partially structured state of the protein is observed. This state shows no structural cooperativity against further denaturation and is characterized by an ellipticity in the far-UV CD greater than that of the native protein. Near-UV CD intensity is dramatically reduced compared with that of the native state, and 1H NMR studies indicate that side-chain interactions are substantially averaged in this denatured state. Solvent proton/deuterium exchange rates for 66 amide hydrogens were measured site-specifically by a combination of amide trapping experiments and 2D 1H NMR. Significant protection from exchange occurs for about 25 backbone amides, the majority of which are located in regions of the protein that are helical in the native enzyme. By contrast, amides located in a second region of the native protein which contains a beta-sheet and one 3(10)-helix as well as a long loop show little protection. This pattern of protection resembles that found in the stable molten globule state of alpha-lactalbumin and in an early kinetic intermediate detected in the refolding of hen lysozyme.
Amino Acid Sequence↗
Human endogenous retroviral element K10 (HERV-K10) encodes a full-length gag homologous 73-kDa protein and a functional protease.
The gag-homologous region of the human endogenous retrovirus K10 (HERV-K10) was amplified by PCR from human genomic DNA and was analyzed by DNA cloning, sequencing, and expression of open reading frames in the prokaryotic pATH expression system. The analysis of genomic DNA of three donors provided evidence that HERV-K10 genes contain an open reading frame of 1966 bp spanning the entire gag-homologous region. In the prokaryotic system the entire reading frame of the HERV-K10 gag gene could be expressed as a fusion protein exhibiting a molecular weight of about 110,000. In addition, when the gag-homologous region and the adjacent HERV-K10 protease gene were prokaryotically expressed, we observed a Gag-protease fusion protein that exhibited specific autoproteolytic activities and processing of HERV-K10 Gag protein. By introducing deletions on the right end of the putative protease gene an autocatalytic site could be localized within 300 bp of the putative HERV-K10 protease gene. For the first time, these results provide evidence that the HERV-K10 encodes a full-length Gag protein and a functional protease.
Base Sequence↗
The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the NifA promoter.
The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed sigma 54-dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60-200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase-sigma 54 complex (E sigma 54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between E sigma 54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase-sigma 54-binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp. Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that E sigma 54 forms a closed complex at DPE1 but not DPE2 and that bound E sigma 54 closely approaches the -15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.
Amino Acid Sequence↗
Core RNA polymerase assists binding of the transcription factor sigma 54 to promoter DNA.
The sigma subunit of bacterial RNA polymerase is necessary for the specific binding of RNA polymerase holoenzyme to promoter DNA. Promoter complexes which form with holoenzyme containing sigma 54 remain as closed complexes unless they are activated by one class of enhancer binding protein. The sigma 54 transcription factor can bind specifically to certain promoter sites in the absence of the core RNA polymerase subunits. This property has allowed demonstration of a new role for core polymerase in transcription, namely that it assists the binding of sigma 54 to promoter DNA. An altered form of sigma 54 with a deletion within the amino-terminal region showed increased affinity for specific DNA-binding sites. Although able to complex with core RNA polymerase the mutant sigma 54 failed to respond to core polymerase in the manner characteristic of the wild-type sigma 54 by altering its footprint. This result indicates that sigma 54 has a latent DNA-binding activity which is revealed by core RNA polymerase, and possibly involves a change in sigma 54 conformation. Promoter complexes which formed with sigma 54-holoenzyme appeared to be qualitatively different, depending upon the target promoter sequence, suggesting that different activatable complexes form at different promoter sequences.
Bacterial Proteins↗
Specific binding of the transcription factor sigma-54 to promoter DNA.
A central event in transcription is the assembly on DNA of specific complexes near the initiation sites for RNA synthesis. Activation of transcription by one class of enhancer-binding proteins requires an RNA polymerase holoenzyme containing the specialized transcription factor, sigma-54 (sigma 54). We report here that sigma 54 alone specifically binds to promoter DNA and is responsible for many of the close contacts between RNA polymerase holoenzyme and promoter DNA, a property proposed for the major sigma 70 protein family. Binding of sigma 54 to promoter DNA is not equivalent to that of holoenzyme suggesting that there is a constraint on sigma 54 conformation when bound with core RNA polymerase. Footprints indicate sigma 54 is at the leading edge of DNA-bound holoenzyme. Like the holoenzyme, sigma 54-binding to promoter DNA does not result in DNA strand separation. Instead the specific DNA-binding activity of sigma 54 assists assembly of a closed promoter complex. This complex can be isomerized to the open (DNA melted) complex by activator protein, but promoter-bound sigma 54 alone cannot be induced to melt DNA. The pathway leading to productive transcription is similar to that proposed for eukaryotic RNA polymerase II systems.
Base Sequence↗
Central domain of the positive control protein NifA and its role in transcriptional activation.
The positive control protein NifA of Klebsiella pneumoniae activates transcription by RNA polymerase containing sigma 54 by catalysing open promoter complex formation. We show that the integrity of the putative ATP-binding pocket in the central domain of NifA is necessary for the positive control function of NifA, but is not required for DNA-binding or recognition of NifA by NifL. The inactive mutant NifA proteins are trans dominant to wild-type NifA and are unable to catalyse formation of open promoter complexes irrespective of whether a closed promoter complex at the nifH promoter has preformed. Formation of the closed complex results in a DNA structural distortion adjacent to the DNA region melted in the open promoter complex. This distortion lies at the leading edge of the E sigma 54 footprint. Although unable to catalyse open complex formation, some mutant NifAs altered the chemical reactivity of the distorted base-pair indicating that they retain the ability to recognize the closed promoter complex. The activation phenotype of partially active NifA molecules was sensitive to promoter sequences known to influence closed complex formation, indicating differences in (1) the susceptibility of the closed complexes towards activation and (2) their requirements for NifA during activation.
Amino Acid Sequence↗
Vibrational spectra of hydrogen on diamond C(111)-(1 x 1).
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Journal Article↗
Hydrogen exchange in native and denatured states of hen egg-white lysozyme.
The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.
Amides↗
Expression of the HIV-1 Nef protein in the baculovirus system: investigation of anti-Nef antibodies response in human sera and subcellular localization of Nef.
The nef gene of HIV-1 was expressed in insect cells using the eucaryotic baculovirus system. The recombinant Nef protein frequently reacted with seropositive sera of HIV-1 and HIV-2 infected patients. Anti-Nef antibodies in HIV-1 seronegative high risk groups individuals were only occasionally seen. Confocal laser scanning microscopy demonstrated that Nef is present both in the cytoplasm and in the nucleus, indicating that Nef might directly function on gene expression.
Baculoviridae↗
Expression of Epstein-Barr virus membrane antigen gp350/220 in E. coli and in insect cells.
The Epstein-Barr virus open reading frame BLLF1 encodes the major envelope glycoproteins gp350 and gp220. Fragments of the gp350/220 gene were expressed in Escherichia coli in order to define regions of the polypeptide chain reacting with human sera. The C-terminal half of the protein was sufficient for recognition by all VCA-positive sera tested. A membrane anchor truncated version of gp350/220 was expressed in insect cells using the baculovirus system. Proteins of different sizes were specifically detected in the cells while a glycosylated 220-kDa protein was secreted. The insect cells were tested for their suitability as tools for performing monospecific immunofluorescence.
Animals↗
Activator-independent formation of a closed complex between sigma 54-holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae.
The alternative sigma factor sigma 54 is required for transcription of nitrogen fixation genes in Klebsiella pneumoniae and other diazotrophs. The nif genes, and other E sigma 54-dependent genes whose products are necessary for a wide range of processes, are postively regulated. A unifying model that is well supported by studies on nif and other nitrogen-regulated (ntr) genes includes the central tenet that sigma 54 confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator-independent complex between E sigma 54 and the NifA-dependent K. pneumoniae nifH and nifU promoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the E sigma 54 recognition sequence.
Base Sequence↗
Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein.
Expression of the Pseudomonas putida TOL plasmid upper pathway operon requires a promoter that belongs to the -12/-24 class. Stimulation of transcription from this promoter is positively controlled by the effector-activated XylR protein and requires a form of RNA-polymerase holoenzyme containing the RpoN-encoded sigma factor, sigma 54. XylR-dependent stimulation of transcription from the Pseudomonas TOL upper pathway promoter was examined using deletions, insertions, and in vivo dimethyl sulfate footprinting. Two upstream activator sequences were identified in the -160 (UAS1) and -130 (UAS2) regions. Deletion of these two regions abolished transcription activation, although conservation of the UAS2 element alone allowed limited transcription stimulation. Separation of UAS1 from UAS2 by half a turn or a full turn significantly reduced XylR stimulation of transcription from the upper pathway operon promoter. An inverted repeated ATTTGN2CAAAT (where N is any nucleoside), which most likely represented the XylR recognition sequence, was identified. Binding of XylR was observed in vivo in the absence of effector, but changes in the binding pattern were induced in the presence of m-methylbenzyl alcohol, a XylR effector. In vivo footprinting analysis revealed that changes in the methylation pattern of G and T also occurred in the -50 to -90 region, which is probably occupied by integration host factor (IHF) protein. IHF was required for maximal expression from the TOL upper pathway operon promoter in Escherichia coli. Separation of the IHF site from UAS2 by a full helix turn did not significantly affect stimulation of transcription, which is consistent with this region playing a conformational role, rather than a regulatory one, in promoter function.
Autoradiography↗
Organization and function of binding sites for the transcriptional activator NifA in the Klebsiella pneumoniae nifE and nifU promoters.
The interaction of the Klebsiella pneumoniae NifA protein, a sigma 54-dependent activator, with the nifE and nifU promoters was analysed. At these promoters NifA established contacts in addition to those predicted by the minimal formulation NifA binding site (5'-TGT-N10-ACA). The positions of the contacts indicate that bound NifA molecules could assemble to form an oligomer. At both promoters contacts with NifA are made predominantly on one face of the DNA helix, and all contacts appear necessary for full activation by NifA. The close contacts made by NifA appear to be made by the DNA-binding domain of NifA. This domain shows specific DNA-binding activity in vitro. The binding of NifA to one site in the nifU promoter depends upon occupancy of additional upstream sequences by NifA. At the nifE promoter NifA binds adjacent to an integration host factor (IHF) binding site, but in contrast to results obtained with the nifU promoter IHF does not diminish nifE promoter occupancy by NifA. The IHF requirement for efficient in vivo activation of the nifU promoter by NifA was greater than that of the nifE promoter. Accordingly, the affinity of IHF for the nifU promoter is higher than for the nifE promoter. Amongst promoters utilizing the sigma 54 holoenzyme, the nifE promoter appears somewhat atypical in having the activator bound at around position -74 rather than the usual 100 base-pairs or more upstream from the transcription start site.
Bacterial Proteins↗