Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human upstream binding factor (hUBF) belonging to a family of protein containing DNA binding domain-HMG box, is important in the activation of rRNA gene transcription. It contains six tandemly arranged HMG box domains, each of which is thought to be as a basic architectural unit in the interaction of DNA and protein. Here the DNA binding domain of hUBF HMG box 1 was cloned and heterologously expressed in Escherichia coli. Through a single purification step using a Ni2+-chelating column, the highly purified recombinant protein could be obtained. This recombinant protein contains 99 amino acids with a hexahistidine tag added to the C-terminus. It was expressed as a monomer, which was determined by gel filtration. Circular dischroism studies show that it comprises approximately 54.3% alpha-helix and 43.6% random coil at pH 7. This result is in good agreement with that of FTIR, which are 59.9% alpha-helix and 40.1% random coil. There is no obvious change for the secondary structure of the recombinant protein as increasing pH from 5.0 to 12.0. But denaturation occurs at pH 3.0. Like many HMG box domains that were found in other proteins, it could bind to four-way DNA junction, a putative intermediate in DNA recombination, in a structure-specific manner. Magnesium ion has no effect on this binding activity, which is determined by both gel mobility shift assays and surface plasmon resonance (SPR). Since Mg2+ is present in the nucleus and RNA polymerase I is Mg2+-stimulated, we believe that this property is relevant for hUBF in vivo. SPR research shows that the recombinant hUBF HMG box 1 also has a strong binding ability to a GC-rich fragment within the rRNA gene core promoter.
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PMID:Cloning, expression, secondary structure characterization of HMG box 1 of hUBF from E. coli and its binding to DNA. 1214 55

The 5'-AMP-activated protein kinase (AMPK) plays a critical role in the regulation of cellular energy homeostasis. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). To date, purified AMPK has only been obtained in small, microgram quantities from tissues. Here, we describe an expression and purification system for production of functional AMPK in Escherichia coli. A plasmid carrying all three subunits of AMPK (alpha1, beta1, and gamma1) for T7 RNA polymerase-driven transcription of a single tricistronic messenger was constructed, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. AMPK was purified from the bacterial lysates by single-step nickel-ion chromatography, utilizing a poly-histidine tag fused to the N-terminus of the alpha-subunit. The recombinant AMPK complex was monodisperse, as shown by gel filtration chromatography with elution of a single peak at a Stokes radius of 52A. Bacterially expressed AMPK was entirely inactive, yet it could be activated by upstream kinase in the presence of AMP. Sufficient quantities of purified functional AMPK should prove to be an invaluable tool to solve many of the pertinent questions about its molecular structure and function, in particular facilitating protein crystallization for X-ray structure analysis.
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PMID:Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. 1288 Jul 72

A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase; EC 2.3.1.30) was isolated by screening a soybean seedling cDNA library with a (32)P-labeled expressed sequence tag. Nucleotide sequence analysis of the isolated cDNA revealed a single open-reading frame of 858 base pairs encoding a 30-kDa polypeptide. The deduced amino acid sequence of soybean SATase revealed significant homology with other plant SATases. Analysis of genomic DNA by Southern blotting indicated that SATase is encoded by a small gene family. The authenticity of the isolated SATase cDNA was confirmed by the expression of the cDNA in an Escherichia coli cysteine-auxotrophic mutant resulting in the growth of the mutant in minimal medium without cysteine. Expression of soybean SATase in E. coli resulted in the production of a 34-kDa protein that was subsequently purified by nickel-affinity column chromatography. The purified protein exhibited SATase activity, indicating that the E. coli-expressed protein is a functionally active SATase. The recombinant soybean SATase was inhibited by l-cysteine, the end product of the cysteine biosynthetic pathway. Antibodies raised against the recombinant soybean SATase cross-reacted with a 34-kDa protein from Arabidopsis leaves, but failed to detect any proteins from soybean leaves and seeds. Reverse transcriptase-polymerase chain reaction analysis indicated that SATase mRNA was expressed at low levels during soybean seed development. In comparison to Arabidopsis leaves, the SATase activity was several-fold lower in soybean leaves and seeds, suggesting that SATase is a low-abundance enzyme.
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PMID:Sulfur assimilation in soybean ( Glycine max [L.] Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase. 1458 54

Ureases are multisubunit enzymes requiring Ni(2+) for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni(2+) uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni(2+) uptake in S. salivarius, regions 3' to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate (63)Ni(2+) during growth. Supplementation of the growth medium with NiCl(2) at concentrations as low as 2.5 micro M partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni(2+) was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni(2+)-specific ATP-binding cassette transporter.
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PMID:Identification and characterization of the nickel uptake system for urease biogenesis in Streptococcus salivarius 57.I. 1461 41

The mitochondrial RNA polymerase (mtRNAP) from Saccharomyces cerevisiae (yeast) is composed of two nuclear encoded proteins, the core RNA polymerase (Rpo41) and the mitochondrial transcription factor (Mtf1). Although Rpo41 is strikingly similar to the single subunit RNAPs from the T7 and T3 bacteriophage (T7RNAP), the core mtRNAP requires Mtf1 for accurate transcription from a linear promoter-containing DNA template, while T7RNAP does not require any other additional factors for promoter selectivity. The fact that the mtRNAP requires an additional promoter utilization factor makes it an excellent model system for the analysis of the transitions that occur during transcription initiation. However, large-scale purification of the 153 kDa Rpo41 has only been reported from yeast cells, or as a recombinant from baculovirus, both sources requiring extensive purification with poor yields. We have developed a His-tagged Rpo41 expression construct suitable for rapid purification of large amounts of soluble Rpo41 from bacterial cells. Transcriptionally active forms of both wild type and point mutants of Rpo41 can be purified by a combination of batch ion exchange chromatography to remove nucleic acids and nickel affinity chromatography. An additional advantage of the isolation of Rpo41 from bacterial cells is the absence of its associated specificity factor Mtf1. This allows analysis of combinations of mutant forms of both components of the mtRNAP holoenzyme.
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PMID:Expression and purification of wild type and mutant forms of the yeast mitochondrial core RNA polymerase, Rpo41. 1503 75

The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription.
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PMID:Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5. 1517 4

We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.
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PMID:Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12. 1534 66

Immobilization and imaging of protein molecules and protein-DNA complexes on a Langmuir-Blodgett (LB) substrate have been explored here. We have prepared a nickel-arachidate (NiA) monolayer and characterized it through pressure-area isotherm on a LB trough. Recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the Ni-arachidate monolayer through a Ni(II)-histidine interaction. A single molecule of RNA polymerase could be seen through intermittent-contact atomic force microscopy (AFM). Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specific DNA molecules from the subphase in an oriented fashion. On the other hand, preformed RNA polymerase Ni(II)-arachidate monolayers bound DNA haphazardly when no surface pressure was employed.
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PMID:Langmuir monolayer as a tool toward visualization of a specific DNA-protein complex. 1626 35

We describe a vector-based system for the production of recombinant Bacillus subtilis RNA polymerase. The recombinant enzyme is C-terminally tagged with nine consecutive histidine residues resulting in about 90% pure enzyme in a single nickel-affinity purification step. The vectors permitted production of recombinant enzyme lacking an omega subunit or containing either the omega(1) (YkzG) or omega(2) (YloH) subunits. In transcription time-course assays all of the recombinant enzymes exhibited identical activity to native RNAP. The modular assembly of the artificial RNA polymerase operon permits ready mutation of any subunit and incorporation into the recombinant enzyme, which will enable new functional/structural studies with this enzyme.
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PMID:Overproduction and purification of recombinant Bacillus subtilis RNA polymerase. 1828 74

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.
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PMID:Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase. 2045 17


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