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Jan Barciszewski - One of the best experts on this subject based on the ideXlab platform.
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structural requirements for conserved arg52 residue for interaction of the human immunodeficiency virus type 1 trans activation responsive element with trans activator of transcription protein 49 57 capillary electrophoresis mobility shift assay
Journal of Chromatography A, 2002Co-Authors: Piotr Mucha, Agnieszka Szyk, Piotr Rekowski, Jan BarciszewskiAbstract:A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualiTative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested 52 that a single, conserved arginine residue (presumably Arg ) within the arginine-rich region (ARR) of Tat plays the major 52 role for the Tat-TAR recognition. To study structural requirements of the Arg position, Tat(49-57)-NH analogues 2 52 substituted with nonencoded amino acids at the Arg position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49-57) a significant shift of migration time of TAR from 18.66 min (RSD 51.4%) to 20.12 min 52 (RSD52.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg 52 (L-Arg → Cit, → Orn, → Arg(NO ), → Arg(Me )) strongly disrupted or abolished the TAR-Tat peptide interaction. 22 52 Enantiomeric substitution, L-Arg → D-Arg was the only one which notably promoted TAR-Tat peptide interaction. The 52 results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg plays a crucial role for TAR-Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA-peptide complex formation. The methodology presented should be generally applicable to study RNA- peptide (protein) interaction. 2002 Elsevier Science B.V. All rights reserved.
Tsafi Peery - One of the best experts on this subject based on the ideXlab platform.
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human and rodent transcription elongation factor p tefb interactions with human immunodeficiency virus type 1 Tat and carboxy terminal domain substrate
Journal of Virology, 1999Co-Authors: Y Ramanathan, Syed M Reza, Tara M Young, Michael B Mathews, Tsafi PeeryAbstract:The human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat, the transactivator of transcription, dramatically increases the production of viral RNA (for a review, see reference 26). This effect is dependent on the transactivation response element (TAR) located downstream of the promoter in the HIV long terminal repeat (LTR). TAR is an RNA element, and TAR RNA binds directly to Tat (17, 72). Although Tat may also exert a stimulatory effect on transcriptional initiation, its predominant effect in vivo appears to be at the level of transcriptional elongation (42, 46, 47). In one model, Tat serves to promote the formation of highly processive RNA polymerase complexes at the HIV LTR. In the absence of Tat, HIV-directed transcripts tend to terminate prematurely at apparently random sites (45). This model is supported by experiments conducted with cell-free transcription systems (28, 44, 50, 51). A unique feature of the stimulation of HIV-directed transcription in vitro is the observation that it is preferentially inhibited by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), an adenosine analogue that targets RNA polymerase II (pol II)-mediated elongation in vitro and in vivo (5, 51). Efforts to uncover the mechanism of Tat action have included extensive searches for cellular components that interact with Tat in vivo and in vitro. This approach disclosed structural and functional interactions with several general transcription factors as well as other proteins with as-yet-unknown roles in transcription (8, 23, 38, 41, 43). Outstanding among these Tat-interacting proteins is a novel Tat-associated protein kinase, TAK, found by Herrmann and Rice (35, 36). This kinase is able to phosphorylate the carboxy-terminal repeat domain (CTD) of pol II and is highly sensitive to DRB. The mammalian CTD consists of a series of 52 heptad repeats, YSPTSPS, located on the large subunit of pol II. It participates in gene expression at several levels and appears to coordinate the machinery of transcription and posttranscriptional RNA processing (37, 55, 57). The CTD can be extensively phosphorylated at multiple sites, predominantly on serine but also on threonine and tyrosine residues (11). Its phosphorylation plays a dominant role in transcription regulation: the hyperphosphorylated form of the large subunit (IIo) is associated with transcription elongation complexes, while only the hypomodified form (IIa) can assemble into the preinitiation complex (60). During the transition from initiation to elongation, or soon after, IIa is converted to IIo (10). The role of the CTD in transcription is not entirely understood, however, and it is possible that more than one round of phosphorylation is required, perhaps with intervening dephosphorylation. The CTD is essential for cell viability in yeast and for transcription from TatA-less promoters in vitro but is nonessential for TatA-containing promoters in vitro (1, 48, 59, 70). It is required for Tat transactivation (7, 61, 75). At least 10 kinases which can phosphorylate the CTD in vitro are known, and 3 of them have an established connection with the transcription machinery. These are CDK7/cyclin H, subunits of the CDK-activating kinase (CAK) complex, which is a subassembly of transcription factor TFIIH; CDK8/cyclin C, a component of the pol II holoenzyme; and CDK9/cyclin T, now identified as components of the positive transcription elongation factor P-TEFb. P-TEFb was first recognized in Drosophila Kc cell extract as an activity needed to overcome abortive elongation and allow formation of long transcripts (53). P-TEFb is distinguished from other transcription factors by its sensitivity to very low doses of DRB (52). It differs from TFIIH, which exhibits less sensitivity to DRB, in that TFIIH functions in initiation and promoter clearance while P-TEFb has the attributes of an elongation factor (18, 53, 54, 62). The possibility that TAK corresponds to human P-TEFb was suggested by the functional similarity between the stimulation of elongation by Tat and the shift to productive elongation by P-TEFb. Circumstantial support for this inference was provided by the observations that both P-TEFb and TAK are CTD kinases and that low concentrations of DRB inhibit P-TEFb, TAK, and the Tat effect (35, 51–53). Conclusive evidence came when Zhu et al. (80) cloned the catalytic subunit of Drosophila P-TEFb and showed that its human homologue (PITALRE) is identical to the kinase subunit of TAK. PITALRE, now called CDK9 (67, 73), was first cloned by Grana et al. (29) as a CDC2-related kinase of unknown function. Most of the kinases in this family have cyclin-regulating subunits, and the cyclin partners of human and Drosophila CDK9 were recently cloned and studied (66). The human cyclins T1, T2a, and T2b were all identified as functional partners of CDK9, and almost all of the CDK9 in HeLa nuclear extracts is associated with either cyclin T1 or T2 (67). Cyclin T1 can also bind directly to the activation domain of Tat (73). Several additional proteins are also associated with CDK9 in immunocomplexes (24, 78, 80), suggesting that human P-TEFb is a multiprotein complex or that there are other CDK9-containing complexes in addition to P-TEFb. It is also likely that Tat binds other factors in addition to CDK9/cyclin T (79). The gene for human cyclin T1 maps to chromosome 12, and this chromosome is necessary for a strong Tat effect on transcription from the HIV LTR in mouse and Chinese hamster ovary (CHO) cells (73). Transfection of CHO and mouse cells with the cyclin T1 gene overcomes the poor Tat response that was previously noticed in these cells (73). The species specificity of the Tat response is also partially complemented in mouse cells harboring human chromosome 6, but the factor(s) responsible is unknown. Our study addresses the interaction of CDK9/cyclin T1 with Tat and its CTD substrate. We demonstrate that human Tat-associated P-TEFb contains cyclin T1 and that rodent P-TEFb is deficient in binding to Tat. Human cyclin T1 as part of the P-TEFb complex in CHO cells complements this deficiency and thus contributes to HIV-1 species specificity. We also show that human TAK is part of a large complex that phosphorylates serine 5 in the CTD heptad and is itself phosphorylated on serine and threonine residues. Our data suggest that TAK and CAK differentially phosphorylate the CTD substrate.
Piotr Mucha - One of the best experts on this subject based on the ideXlab platform.
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structural requirements for conserved arg52 residue for interaction of the human immunodeficiency virus type 1 trans activation responsive element with trans activator of transcription protein 49 57 capillary electrophoresis mobility shift assay
Journal of Chromatography A, 2002Co-Authors: Piotr Mucha, Agnieszka Szyk, Piotr Rekowski, Jan BarciszewskiAbstract:A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualiTative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested 52 that a single, conserved arginine residue (presumably Arg ) within the arginine-rich region (ARR) of Tat plays the major 52 role for the Tat-TAR recognition. To study structural requirements of the Arg position, Tat(49-57)-NH analogues 2 52 substituted with nonencoded amino acids at the Arg position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49-57) a significant shift of migration time of TAR from 18.66 min (RSD 51.4%) to 20.12 min 52 (RSD52.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg 52 (L-Arg → Cit, → Orn, → Arg(NO ), → Arg(Me )) strongly disrupted or abolished the TAR-Tat peptide interaction. 22 52 Enantiomeric substitution, L-Arg → D-Arg was the only one which notably promoted TAR-Tat peptide interaction. The 52 results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg plays a crucial role for TAR-Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA-peptide complex formation. The methodology presented should be generally applicable to study RNA- peptide (protein) interaction. 2002 Elsevier Science B.V. All rights reserved.
Y Ramanathan - One of the best experts on this subject based on the ideXlab platform.
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human and rodent transcription elongation factor p tefb interactions with human immunodeficiency virus type 1 Tat and carboxy terminal domain substrate
Journal of Virology, 1999Co-Authors: Y Ramanathan, Syed M Reza, Tara M Young, Michael B Mathews, Tsafi PeeryAbstract:The human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat, the transactivator of transcription, dramatically increases the production of viral RNA (for a review, see reference 26). This effect is dependent on the transactivation response element (TAR) located downstream of the promoter in the HIV long terminal repeat (LTR). TAR is an RNA element, and TAR RNA binds directly to Tat (17, 72). Although Tat may also exert a stimulatory effect on transcriptional initiation, its predominant effect in vivo appears to be at the level of transcriptional elongation (42, 46, 47). In one model, Tat serves to promote the formation of highly processive RNA polymerase complexes at the HIV LTR. In the absence of Tat, HIV-directed transcripts tend to terminate prematurely at apparently random sites (45). This model is supported by experiments conducted with cell-free transcription systems (28, 44, 50, 51). A unique feature of the stimulation of HIV-directed transcription in vitro is the observation that it is preferentially inhibited by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), an adenosine analogue that targets RNA polymerase II (pol II)-mediated elongation in vitro and in vivo (5, 51). Efforts to uncover the mechanism of Tat action have included extensive searches for cellular components that interact with Tat in vivo and in vitro. This approach disclosed structural and functional interactions with several general transcription factors as well as other proteins with as-yet-unknown roles in transcription (8, 23, 38, 41, 43). Outstanding among these Tat-interacting proteins is a novel Tat-associated protein kinase, TAK, found by Herrmann and Rice (35, 36). This kinase is able to phosphorylate the carboxy-terminal repeat domain (CTD) of pol II and is highly sensitive to DRB. The mammalian CTD consists of a series of 52 heptad repeats, YSPTSPS, located on the large subunit of pol II. It participates in gene expression at several levels and appears to coordinate the machinery of transcription and posttranscriptional RNA processing (37, 55, 57). The CTD can be extensively phosphorylated at multiple sites, predominantly on serine but also on threonine and tyrosine residues (11). Its phosphorylation plays a dominant role in transcription regulation: the hyperphosphorylated form of the large subunit (IIo) is associated with transcription elongation complexes, while only the hypomodified form (IIa) can assemble into the preinitiation complex (60). During the transition from initiation to elongation, or soon after, IIa is converted to IIo (10). The role of the CTD in transcription is not entirely understood, however, and it is possible that more than one round of phosphorylation is required, perhaps with intervening dephosphorylation. The CTD is essential for cell viability in yeast and for transcription from TatA-less promoters in vitro but is nonessential for TatA-containing promoters in vitro (1, 48, 59, 70). It is required for Tat transactivation (7, 61, 75). At least 10 kinases which can phosphorylate the CTD in vitro are known, and 3 of them have an established connection with the transcription machinery. These are CDK7/cyclin H, subunits of the CDK-activating kinase (CAK) complex, which is a subassembly of transcription factor TFIIH; CDK8/cyclin C, a component of the pol II holoenzyme; and CDK9/cyclin T, now identified as components of the positive transcription elongation factor P-TEFb. P-TEFb was first recognized in Drosophila Kc cell extract as an activity needed to overcome abortive elongation and allow formation of long transcripts (53). P-TEFb is distinguished from other transcription factors by its sensitivity to very low doses of DRB (52). It differs from TFIIH, which exhibits less sensitivity to DRB, in that TFIIH functions in initiation and promoter clearance while P-TEFb has the attributes of an elongation factor (18, 53, 54, 62). The possibility that TAK corresponds to human P-TEFb was suggested by the functional similarity between the stimulation of elongation by Tat and the shift to productive elongation by P-TEFb. Circumstantial support for this inference was provided by the observations that both P-TEFb and TAK are CTD kinases and that low concentrations of DRB inhibit P-TEFb, TAK, and the Tat effect (35, 51–53). Conclusive evidence came when Zhu et al. (80) cloned the catalytic subunit of Drosophila P-TEFb and showed that its human homologue (PITALRE) is identical to the kinase subunit of TAK. PITALRE, now called CDK9 (67, 73), was first cloned by Grana et al. (29) as a CDC2-related kinase of unknown function. Most of the kinases in this family have cyclin-regulating subunits, and the cyclin partners of human and Drosophila CDK9 were recently cloned and studied (66). The human cyclins T1, T2a, and T2b were all identified as functional partners of CDK9, and almost all of the CDK9 in HeLa nuclear extracts is associated with either cyclin T1 or T2 (67). Cyclin T1 can also bind directly to the activation domain of Tat (73). Several additional proteins are also associated with CDK9 in immunocomplexes (24, 78, 80), suggesting that human P-TEFb is a multiprotein complex or that there are other CDK9-containing complexes in addition to P-TEFb. It is also likely that Tat binds other factors in addition to CDK9/cyclin T (79). The gene for human cyclin T1 maps to chromosome 12, and this chromosome is necessary for a strong Tat effect on transcription from the HIV LTR in mouse and Chinese hamster ovary (CHO) cells (73). Transfection of CHO and mouse cells with the cyclin T1 gene overcomes the poor Tat response that was previously noticed in these cells (73). The species specificity of the Tat response is also partially complemented in mouse cells harboring human chromosome 6, but the factor(s) responsible is unknown. Our study addresses the interaction of CDK9/cyclin T1 with Tat and its CTD substrate. We demonstrate that human Tat-associated P-TEFb contains cyclin T1 and that rodent P-TEFb is deficient in binding to Tat. Human cyclin T1 as part of the P-TEFb complex in CHO cells complements this deficiency and thus contributes to HIV-1 species specificity. We also show that human TAK is part of a large complex that phosphorylates serine 5 in the CTD heptad and is itself phosphorylated on serine and threonine residues. Our data suggest that TAK and CAK differentially phosphorylate the CTD substrate.
David L Yirrell - One of the best experts on this subject based on the ideXlab platform.
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Tat HIV 1 primary and tertiary structures critical to immune response against non homologous variants
Journal of Biological Chemistry, 2002Co-Authors: Sandrine Opi, Jeanmarie Peloponese, Didier Esquieu, Grant R Campbell, Jean De Mareuil, Anne Walburger, Murielle Solomiac, Catherine Gregoire, Emmanuelle Bouveret, David L YirrellAbstract:Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that muTations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzyme-linked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.
