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)

RNA polymerase II transcripts accumulate within mammalian nuclei at distinct sites and exhibit varying morphology. Certain RNA species are organized in elongated structures, whereas others appear as dot-like concentrations. To analyze the status of the RNA within these accumulations, we investigated the composition of accumulations derived from Epstein-Barr virus (EBV) genes, human papilloma virus 18 (HPV18) open reading frames E6 and E7, as well as heat shock protein 89a (hsp89alpha) and 89beta (hsp89beta) genes. No differential distribution of exon and intron sequences within concentrations of EBV RNA could be observed. Whereas accumulations of hsp89alpha and hsp89beta always coincided with Sm antigen foci, the RNA of EBV and HPV18 never co-localized with these foci. This excludes Sm antigen foci as the only sites of splicing and suggests gene-specific variation in the nuclear localization of transcripts. Two sets of experiments were performed to assess whether transcripts in the RNA accumulations are in statu nascendi or products released from a discrete gene locus. Because RNA transcripts derived from EBV genes, which are located on both ends of the genome, were all distributed along the entire length of the RNA signals, they cannot be derived from a highly decondensed genomic DNA extending throughout elongated RNA accumulations. Furthermore, removal of labeled RNA sequences and subsequent visualization of DNA confirmed the confinement of the genomic sequences to a small subregion of the area occupied by accumulated RNA. Therefore, this study supports the view of RNA accumulations as a stream of molecules that delineate a path from a dot-like gene locus toward the nuclear envelope for export into the cytoplasm.
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PMID:Nuclear RNA accumulations contain released transcripts and exhibit specific distributions with respect to Sm antigen foci. 936 24

Hsp70, the most abundant and conserved heat shock protein, has been described as strongly concentrating in the nucleolus during heat shock. The important metabolic processes that take place in the nucleolus, rDNA transcription, processing, and assembling with ribosomal proteins, and the nucleolar architecture itself are very sensitive to temperature changes. In this work, we have analyzed in detail the nucleolar changes, in structure and activity, induced by temperature in Chironomus thummi salivary gland cells and the fine subnucleolar localization of Hsp70 during heat shock. The optimum temperature chosen to induce the heat shock response was 35 degrees C. Under these conditions transcription of heat shock genes, inactivation of previously active genes and maximum synthesis of Hsps take place, while survival of larvae and recovery were ensured. After 1 h at 35 degrees C, nucleoli change from a uniform control pattern to a segregated pattern of nucleolar components that can be observed even at the light microscopic level. The dense fibrillar component (DFC) and the granular component appeared perfectly differentiated and spatially separated, the former occupying mainly the central inner region surrounded by a rim of granular component. Hsp70 was specifically localized within the DFC upon heat shock as shown by immunolocalization by both light and electron microscopy. Pulse labeling with [3H]uridine proves that rRNA transcription continues during heat shock. The pattern of Hsp70 distribution within the nucleolus correlates with that of newly produced rRNA transcripts. Hsp70 also colocalizes with RNA polymerase I, both being restricted to the DFC. These data show that the DFC seems to be the intranucleolar target for Hsp70 in heat-shocked cells. We discuss these results in relation to the possible function of Hsp70 in the first steps of preribosome synthesis.
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PMID:Specific intranucleolar distribution of Hsp70 during heat shock in polytene cells. 936 19

The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E. coli using the bacteriophage T7 RNA polymerase-based gene expression system. Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography. R-hsp70 was shown to be monomeric and free of its structurally similar E. coli counterpart, DnaK. In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP. The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70. Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.
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PMID:Human stress protein hsp70: overexpression in E coli, purification and characterization. 963 84

The possible functionality of short interspersed elements (SINEs) is investigated by assaying the effects of physiological stress on their RNA polymerase-III-directed transcriptional expression in vivo. B2 RNA is expressed at moderately high levels in all mouse tissues investigated, namely liver, spleen, kidney and testis. B1 RNA is expressed in testis but is nearly undetectable in the other tissues. Following hyperthermic shock, the amounts of B1 and B2 SINE RNAs transiently increase in all tissues by as much as 40-fold in certain cases. The kinetics of these increases resemble those of heat shock protein mRNAs. An acute dose of ethanol also transiently increases the abundance of B1 and B2 RNA in liver, showing that other physiological stresses increase SINE RNA expression. The constitutive expression of B2 RNA in all tissues and tissue-specific differences in expression of B1 RNA imply that these transcripts serve a normal physiological function(s). Moreover, increased SINE RNA expression is a vital response to stress and by the criterion of their inducibility, mammalian SINEs behave like regulated cell stress genes.
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PMID:Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo. 1054 39

A large variety of stress conditions including physicochemical factors induce the synthesis of more than 20 heat shock proteins (HSPs). In E. coli, the heat shock response to temperature upshift from 30 to 42 degrees C consists of the rapid induction of these HSPs, followed by an adaptation period where the rate of HSP synthesis decreases to reach a new steady-state level. Major HSPs are molecular chaperones, including DnaK, DnaJ and GrpE, and GroEL and GroES, and proteases. They constitute the two major chaperone systems of E. coli (15-20% of total protein at 46 degrees C). They are important for cell survival, since they play a role in preventing aggregation and refolding proteins. The E. coli heat shock response is positively controlled at the transcriptional level by the product of the rpoH gene, the heat shock promoter-specific sigma32 subunit of RNA polymerase. Because of its rapid turn-over, the cellular concentration of sigma32 is very low under steady-state conditions (10-30 copies/cell at 30 degrees C) and is limiting for heat shock gene transcription. The heat shock response is induced as a consequence of a rapid increase in sigma32 levels and stimulation of sigma32 activity. The shut off of the response occurs as a consequence of declining sigma32 levels and inhibition of sigma32 activity. Stress-dependent changes in heat shock gene expression are mediated by the antagonistic action of sigma32 and negative modulators which act upon sigma32. These modulators are the DnaK chaperone system which inactivate sigma32 by direct association and mediate its degradation by proteases. Degradation of sigma32 is mediated mainly by FtsH (HflB), an ATP-dependent metallo-protease associated with the inner membrane. There is increasing evidence that the sequestration of the DnaK chaperone system through binding to misfolded proteins is a direct determinant of the modulation of the heat shock genes expression. A central open question is the identity of the binding sites within sigma32 for DnaK, DnaJ, FtsH and the RNA polymerase, and the functional interplay between these sites. We have studied the role of two distinct regions of sigma32 in its activity and stability control: region C and the C-terminal part. Both regions are involved in RNA polymerase binding.
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PMID:The heat shock response of Escherichia coli. 1082 72

Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the alpha, beta, and beta' subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val(-4)-Phe(-3)-Asn(-2)-Ala(-1) (downward arrow)Cys(+1) was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31(LipL45). Membrane fractionation studies demonstrated that P31(LipL45) is a peripheral membrane protein. Finally, we found that P31(LipL45) levels increased as Leptospira entered the stationary phase, indicating that P31(LipL45) levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.
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PMID:Novel 45-kilodalton leptospiral protein that is processed to a 31-kilodalton growth-phase-regulated peripheral membrane protein. 1174 98

The 27 kDa heat shock protein (hsp27) is expressed in keratinocytes in a differentiation-related pattern. Keratinocyte differentiation involves a coordinated program of expression and interaction of specific differentiation-related genes and proteins. To investigate the functional role of hsp27 in these processes we used a differential display approach to identify genes that might be regulated by the expression of hsp27 in human keratinocytes. mRNA was extracted from the human squamous carcinoma cell line A431 and a subclone stably transfected with human hsp27. Reverse transcriptase differential display polymerase chain reaction was performed using one base anchored oligo-dT and arbitrary primers. Differentially expressed genes were confirmed by northern blot analysis and further characterized by sequencing. Their expression in human skin and other tissues was investigated by northern blot and in situ hybridization. Out of five fragments detected with the initial reverse transcriptase differential display polymerase chain reaction screen one could be confirmed by northern blot to be downregulated in hsp27-overexpressing A431. This mRNA (G24) is not only downregulated by overexpression of hsp27 in A431 but also during differentiation in normal human keratinocytes in culture and in situ, situations where hsp27 is known to be induced. According to sequence analysis G24 represents a novel gene that does not code for a protein and thus might belong to the growing family of noncoding RNAs. These results not only demonstrate for the first time that overexpression of hsp27 by gene transfer is associated with regulation of gene expression but also reveal a novel differentiation-associated gene in human keratinocytes.
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PMID:Differential expression of a novel gene in response to hsp27 and cell differentiation in human keratinocytes. 1216 38

Archaea have a eukaryotic type of transcriptional machinery containing homologues of the transcription factors TATA-binding protein (TBP) and TFIIB (TFB) and a pol II type of RNA polymerase, whereas transcriptional regulators identified in archaeal genomes have bacterial counterparts. We describe here a novel regulator of heat shock response, Phr, from the hyperthermophilic archaeon Pyrococcus furiosus that is conserved among Euryarchaeota. The protein specifically inhibited cell-free transcription of its own gene and from promoters of a small heat shock protein, Hsp20, and of an AAA(+) ATPase. Inhibition of transcription was brought about by abrogating RNA polymerase recruitment to the TBP/TFB promoter complex. Phr bound to a 29-bp DNA sequence overlapping the transcription start site. Three sequences conserved in the binding sites of Phr, TTTA at -10, TGGTAA at the transcription start site, and AAAA at position +10, were required for Phr binding and are proposed as consensus regulatory sequences of Pyrococcus heat shock promoters. Shifting the growth temperature from 95 to 103 degrees C caused a dramatic increase of mRNA levels for the aaa(+) atpase and phr genes, but expression of the Phr protein was only weakly stimulated. Our findings suggest that heat shock response in Archaea is negatively regulated by a mechanism involving binding of Phr to conserved sequences.
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PMID:A novel archaeal transcriptional regulator of heat shock response. 1238 24

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.
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PMID:Analysis of conserved non-rRNA genes of Tropheryma whipplei. 1274 4

Expression of heat shock genes in Gram-negative proteobacteria is positively modulated by the transcriptional regulator RpoH, the sigma(32) subunit of RNA polymerase (RNAP). In this study we investigated the chaperones DnaK/DnaJ and GroES/GroEL as possible modulators of the heat response in Caulobacter crescentus. We have shown that cells overexpressing DnaK show poor induction of heat shock protein (HSP) synthesis, even though sigma(32) levels present a normal transient increase upon heat stress. On the other hand, depletion of DnaK led to higher levels of sigma(32) and increased transcription of HSP genes, at normal growth temperature. In contrast, changes in the amount of GroES/EL had little effect on sigma(32) levels and HSP gene transcription. Despite the strong effect of DnaK levels on the induction phase of the heat shock response, downregulation of HSP synthesis was not affected by changes in the amount this chaperone. Thus, we propose that competition between sigma(32) and sigma(73), the major sigma factor, for the core RNAP could be the most important factor controlling the shut-off of HSP synthesis during recovery phase. In agreement with this hypothesis, we have shown that expression of sigma(73) gene is heat shock inducible.
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PMID:Downregulation of the heat shock response is independent of DnaK and sigma32 levels in Caulobacter crescentus. 1282 48


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