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)

The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated trans-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.
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PMID:HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors. 155 13

Human immunodeficiency virus type 1 (HIV-1) is viable and mitogen inducible in the absence of its binding sites for the inducible transcription factor NF-kappa B. We have investigated alternative mechanisms for induction of HIV-1 transcription. Using transient transfection assays, we found that transcription from an HIV-1 LTR containing mutant kappa B sites was activated 10- to 20-fold in a variety of human cell types by the phorbol ester phorbol myristate acetate (PMA). The promoter elements conferring this inducibility were localized to the region downstream of nucleotide -70, which contains the TATA and TAR elements and binding sites for transcription factors Sp1 and LBP-1. Synthetic promoters containing only Sp1 sites and a TATA element were also induced in transfection experiments as well as in in vitro transcription experiments with T-cell nuclear extracts. Moreover, promoters containing a TATA box in the absence of Sp1 sites or Sp1 sites in the absence of a TATA box were equally inducible in vitro, as was an RNA polymerase III promoter. The activities of RNA polymerases II and III and of the 38-kDa TATA-binding protein transcription factor IID (TFIID), were not induced by PMA, but electrophoretic mobility shift assays revealed a highly inducible protein-DNA complex that interacted specifically with the TATA sequence. This protein-DNA complex appeared to be much larger than that found with the 38-kDa human TFIID expressed in bacteria. Taken together, these data suggest that a component of the general transcription machinery, and possibly a TFIID-associated protein, is induced in T cells by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Alternative pathway for induction of human immunodeficiency virus gene expression: involvement of the general transcription machinery. 189 93

Multiple binding of Tat and nuclear protein(s) to HIV-1 TAR RNA appears to be essential for the Tat-mediated trans-activation. As synthetic Tat-(1-47), which lacks the basic domain and does not bind TAR RNA in vitro, efficiently transactivated HIV-1 LTR in HeLa nuclear extracts, we hypothesized that Tat might trans-activate by interaction with TAR RNA via a host nuclear protein. The role of nuclear proteins in Tat-TAR interaction was examined through evaluation of several synthetic Tat peptides for ability to bind TAR RNA in vitro both in the presence and in the absence of HeLa nuclear proteins. Our data show that both Tat-(1-47) and Tat-(1-86) interact with TAR RNA-bound nuclear proteins, leading to dissociation of the nuclear protein-TAR RNA complexes; the N-terminal sequence of Tat appears to be involved in this interaction. Thus, after binding to TAR RNA, Tat can interact with a proximal TAR-bound nuclear protein and the resulting Tat-nuclear protein complex, now displaced from TAR, may initiate a facile and rapid assembly of the RNA polymerase II transcription complex. This study thus recognizes a novel interaction between Tat and a nuclear protein(s). Here we propose that the interaction of Tat with a nuclear protein(s) occurring on TAR RNA may be one of several steps in the mechanism of Tat-mediated trans-activation of the HIV-1 LTR.
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PMID:Synthetic HIV-1 Tat can dissociate HeLa nuclear protein-TAR RNA complexes in vitro: a novel Tat-nuclear protein interaction. 192 18

In this study we have defined the in vitro requirements for transcriptional regulation of the HIV-2 LTR in response to the HIV-1 and HIV-2 Tat proteins and addressed potential mechanisms of Tat function. HIV-2 contains a duplicated TAR RNA stem-loop structure in contrast to the single stem-loop structure found in HIV-1 TAR RNA. We demonstrated that the HIV-2 proximal TAR RNA stem-loop structure was more important for in vitro transcriptional activation by the HIV-1 and HIV-2 Tat proteins than the distal TAR RNA stem-loop though this downstream TAR element itself was able to confer Tat-responsiveness. The role of the two HIV-2 TAR RNA stem-loop bulge sequences was less critical than the loop sequences for in vitro transcriptional activation by Tat. In addition, we demonstrated that replacing the HIV-2 TATA element with that of HIV-1 markedly reduced the overall level of Tat activation. The role of the Tat-1 and Tat-2 proteins on the synthesis of HIV-1 and HIV-2 promoter proximal and promoter distal transcripts was then investigated. In contrast to the HIV-1 promoter, the HIV-2 promoter generated abundant levels of short transcripts in vitro transcription assays likely due to the structure of its duplicated TAR element. Both Tat-1 and Tat-2 increased the level of transcripts extending to the end of the HIV-1 and HIV-2 TAR elements as well as the level of transcripts extending more than 500 nucleotides from the transcription initiation site. However, the synthesis of transcripts within 30 nucleotides of the HIV-2 LTR transcription initiation site was unchanged in either the presence or absence of Tat while the level of transcripts extending increasing distances from the HIV-2 LTR transcription initiation site were progressively stimulated in the presence of Tat. Though the HIV-1 Tat protein was a stronger inducer of HIV-1 LTR transcription than the HIV-2 Tat protein, we did not detect differences in the binding of these proteins to the HIV-1 and HIV-2 TAR RNAs. This suggested that differences in their transactivation properties may be due to alterations in their association with RNA polymerase II or associated elongation factors. (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tat functions to stimulate the elongation properties of transcription complexes paused by the duplicated TAR RNA element of human immunodeficiency virus 2. 749 Jul 54

The introduction of isotopically enriched nucleotides into NMR quantities of a synthetic 29-mer RNA derived from the HIV-1 TAR element is described. RNA enriched in 13C and/or 15N is produced by a procedure which involves isolation of whole cellular RNA from Escherichia coli, nucleolysis, separation of mononucleotides, chemical or enzymatic pyrophosphorylation, and in vitro transcription by T7 RNA polymerase. Spectral characteristics of each residue type are examined in isolation. 13C chemical shifts provide an alternative method to determine ribose puckers for larger RNAs. Nonprotonated sites such as purine N7 groups can now be monitored through the use of multiple-bond 1H-15N coupling. When applied conservatively, coordinate analysis of chemical shift values should prove valuable for NMR studies of RNA structure and recognition. 1H, 13C, and 15N chemical shift data suggest that TAR residue A35 has an unusual local environment, consistent with extrusion of its base from the terminal loop.
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PMID:Selective isotopic enrichment of synthetic RNA: application to the HIV-1 TAR element. 842 47

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.
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PMID:Identification of a group of cellular cofactors that stimulate the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA. 862 63

Activation of HIV-1 gene expression by the transactivator Tat is dependent on an RNA regulatory element located downstream of the transcription initiation site known as TAR. To characterize cellular factors that bind to TAR RNA and are involved in the regulation of HIV-1 transcription, HeLa nuclear extract was fractionated and RNA gel-retardation analysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA loop binding protein TRP-185, were capable of binding specifically to TAR RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated. TRP-185 is a novel protein of 1621 amino acids which contains a leucine zipper and potentially a novel RNA binding motif. In gel-retardation assays, the binding of both recombinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both the TAR RNA loop and bulge sequences were critical for RNA polymerase II binding, while TRP-185 binding was dependent only on TAR RNA loop sequences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA polymerase II which is stalled upon binding to nascently synthesized TAR RNA during transcriptional elongation.
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PMID:The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA. 884 92

The HIV-1 (human immunodeficiency virus type 1) and HIV-2 Tat proteins increase the level of transcription from their corresponding long terminal repeats. Tat activates transcription likely by interaction with components of the transcriptional initiation and elongation complexes during different stages of the transcription reaction. In the current study, two approaches were used to address the sites at which Tat becomes stably associated with the HIV transcription complex. First, we isolated column purified HIV-1 and HIV-2 transcription complexes that were competent for in vitro transcription and found that wild-type but not mutant Tat protein was specifically associated with this complex. An intact HIV TATA element and the presence of functional TATA-binding protein were necessary for Tat association. In contrast, the HIV-1 and HIV-2 TAR bulge sequences which serve as binding sites for Tat were not required for its association with the HIV preinitiation complex. A second complementary approach using immobilized HIV-1 and HIV-2 templates also demonstrated a functional association of Tat with HIV-1 and HIV-2 preinitiation complexes. Wild-type but not mutant Tat proteins associated with transcription complexes assembled on immobilized HIV-1 and HIV-2 templates and the association of Tat correlated with increases in the level of in vitro transcription. These results indicate that Tat can associate with HIV-1 and HIV-2 transcription complexes prior to the initiation of transcription by RNA polymerase II.
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PMID:Association of Tat with purified HIV-1 and HIV-2 transcription preinitiation complexes. 905 83

A structural motif at the 5' end of human 7S L (srp) RNA that is recognized specifically by cellular proteins has been identified as an efficient activator of RNA polymerase (pol) III transcription in vivo and in vitro. Mutations affecting three double-stranded regions or a tetranucleotide bulge of this RNA motif result in strongly reduced expression rates. However, effective suppression is achieved by compensatory mutations restoring RNA sequence complementarity. This activation of transcription is also observed in the context of another pol III promoter and is position-dependent. The effects observed are reminiscent of the Tat-TAR trans-activation of the human immunodeficiency virus and attribute a novel function to the structure of cellular small stable RNA.
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PMID:Secondary structure of the nascent 7S L RNA mediates efficient transcription by RNA polymerase III. 914 34

Tat stimulates HIV-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR RNA and several putative cellular cofactors to increase the processivity of RNA polymerase II complexes during HIV-1 transcription elongation. Although HIV-1 transactivation by Tat in most cell types requires intact TAR sequences, previous reports demonstrate that Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that kappa B elements within the HIV-1 LTR mediate TAR-independent transactivation by Tat and demonstrate additionally that distinct amino acid residues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87-MG cell lines expressing a transdominant negative mutant of I kappa B alpha, I kappa B alpha delta N, fail to support TAR-independent transactivation by Tat, suggesting that binding of NF-kappa B to kappa B enhancer elements within the HIV-1 LTR is necessary for Tat-mediated transactivation in the absence of TAR. Ribonucleic acid protection analyses of promoter-proximal and -distal transcripts derived from TAR-deleted and TAR-containing HIV-1 LTR reporter constructs in U87-MG cells indicate that the predominant effect of Tat during TAR-independent transactivation occurs at the lavel of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transactivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interaction of Tat with NF-kappa B-binding sites in the HIV-1 LTR.
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PMID:Distinct transcriptional pathways of TAR-dependent and TAR-independent human immunodeficiency virus type-1 transactivation by Tat. 930 36


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