The Mechanism of Release of P-TEFb and HEXIM1 from the 7SK snRNP by Viral and Cellular Activators Includes a Conformational Change in 7SK
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{"title"=>"The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK", "type"=>"journal", "authors"=>[{"first_name"=>"Brian J.", "last_name"=>"Krueger", "scopus_author_id"=>"24076512500"}, {"first_name"=>"Katayoun", "last_name"=>"Varzavand", "scopus_author_id"=>"36115979600"}, {"first_name"=>"Jeffrey J.", "last_name"=>"Cooper", "scopus_author_id"=>"8364480600"}, {"first_name"=>"David H.", "last_name"=>"Price", "scopus_author_id"=>"7403719243"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-77957880315", "sgr"=>"77957880315", "pui"=>"359754365", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"20808803", "doi"=>"10.1371/journal.pone.0012335"}, "id"=>"17cfc60c-98be-34dd-8f2c-ba53fd444651", "abstract"=>"BACKGROUND: The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.\\n\\nCONCLUSIONS/SIGNIFICANCE: Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.", "link"=>"http://www.mendeley.com/research/mechanism-release-ptefb-hexim1-7sk-snrnp-viral-cellular-activators-includes-conformational-change-7s", "reader_count"=>56, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>5, "Researcher"=>6, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>3, "Other"=>2, "Student > Master"=>5, "Student > Bachelor"=>9, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>5, "Researcher"=>6, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>3, "Other"=>2, "Student > Master"=>5, "Student > Bachelor"=>9, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>9, "Agricultural and Biological Sciences"=>36, "Medicine and Dentistry"=>2, "Neuroscience"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemistry"=>6}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>6}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>36}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>9}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"United States"=>1, "United Kingdom"=>2, "France"=>1, "India"=>1}, "group_count"=>3}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/835024"], "description"=>"<p>A) P-TEFb release reactions were performed as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012335#s4\" target=\"_blank\">Materials and Methods</a>. Indicated amounts of Tat were incubated for the indicated times with immunoprecipitated 7SK snRNPs. B) Same as in A except the RNA binding deficient mutant of Tat (Tat R52/53A) was used. C) Same as in A except the P-TEFb binding deficient mutant of Tat (Tat C22G) was used. D) Tat release was quantified from three independent experiments for wild type Tat and the RNA binding mutant and two independent experiments for the P-TEFb binding mutant. Mean values for the percent of Cdk9 left in the complex are plotted and all error bars represent standard error. E) 100 ng of Tat was incubated with the indicated amounts of TAR or 7SK RNA before being added to the 7SK snRNP. F) Same as in E except TatR52/53A was used.</p>", "links"=>[], "tags"=>["p-tefb", "7sk", "snrnp", "hiv"], "article_id"=>505388, "categories"=>["Biochemistry", "Molecular Biology"], "users"=>["Brian J. Krueger", "Katayoun Varzavand", "Jeffrey J. Cooper", "David H. Price"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012335.g001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Release_of_P_TEFb_from_the_7SK_snRNP_by_HIV_Tat_/505388", "title"=>"Release of P-TEFb from the 7SK snRNP by HIV Tat.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:54:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/835569"], "description"=>"<p>P-TEFb is directly extracted from the 7SK snRNP by Tat or Brd4. This leads to the loss of P-TEFb, a destabilization of the 7SK structure resulting in a conformational change in the RNA that causes HEXIM1 to be released from the 7SK snRNP. hnRNP proteins then bind to the region of RNA unmasked by the loss of HEXIM1.</p>", "links"=>[], "tags"=>["p-tefb", "hexim1", "7sk"], "article_id"=>505937, "categories"=>["Biochemistry", "Molecular Biology"], "users"=>["Brian J. Krueger", "Katayoun Varzavand", "Jeffrey J. Cooper", "David H. Price"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012335.g006", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Model_of_P_TEFb_and_HEXIM1_release_from_the_7SK_snRNP_/505937", "title"=>"Model of P-TEFb and HEXIM1 release from the 7SK snRNP.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:57:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/835130"], "description"=>"<p>A) A schematic of Brd4 constructs used. BD1, Bromodomain 1; BD2, Bromodomain 2; ET, Extraterminal domain; H1, H2 and H3, Helical domains 1, 2 and 3. Brd4 1209–1362 contains three helical regions, Brd4 1209–1362 Δ1329–1345 is missing helical region 3 which is required for P-TEFb binding. B) Recombinant protein expressed and purified from <i>E. coli</i>. A contaminating band can be seen in the Brd4 mutant preparation and this was confirmed to be an <i>E. coli</i> protein by Western blot (data not shown), not full length Brd4. M, Marker; 1, Brd4 1209–1362; 2, Brd4 1209–1362 Δ1329–1345. C) Indicated amounts of Brd4 were added into the release reaction for the indicated times. D) Same as in C except the Brd4 mutant missing helical domain 3 was used for the reactions. E) Brd4 release was quantified from three independent experiments. Two independent experiments were done to calculate the mean for the Brd4 helical domain 3 mutant. The y-axis is a measure of percent of Cdk9 left in the complex. All error bars represent standard error.</p>", "links"=>[], "tags"=>["p-tefb", "7sk", "snrnp", "binding"], "article_id"=>505501, "categories"=>["Biochemistry", "Molecular Biology"], "users"=>["Brian J. Krueger", "Katayoun Varzavand", "Jeffrey J. Cooper", "David H. Price"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012335.g002", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Release_of_P_TEFb_from_the_7SK_snRNP_by_the_P_TEFb_binding_domain_of_Brd4_/505501", "title"=>"Release of P-TEFb from the 7SK snRNP by the P-TEFb binding domain of Brd4.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:55:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/835389"], "description"=>"<p>A) HIV-1 TAR RNA stem loop: 35–61 of TAR RNA covering the Tat binding bulge (AUCUG) and the cyclin T1 binding loop (CUGGG). 7SK Alt: Predicted structure of the 1–100 region of 7SK. 7SK W&S: structure of the 1–100 region of 7SK RNA described previously <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012335#pone.0012335-Wassarman1\" target=\"_blank\">[57]</a>. The uracil residues in 7SK RNA are marked. B) and C) CMCT reactivity was determined as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012335#s4\" target=\"_blank\">Materials and Methods</a> using extension of the indicated primers. C, control cells; F, flavopiridol treated cells; L; sequencing ladder showing the position of uracils.</p>", "links"=>[], "tags"=>["p-tefb", "flavopiridol", "cells", "causes", "conformational", "7sk"], "article_id"=>505756, "categories"=>["Biochemistry", "Molecular Biology"], "users"=>["Brian J. Krueger", "Katayoun Varzavand", "Jeffrey J. Cooper", "David H. Price"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012335.g004", "stats"=>{"downloads"=>1, "page_views"=>28, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Release_of_P_TEFb_by_flavopiridol_treatment_of_cells_causes_a_conformational_change_in_7SK_snRNA_/505756", "title"=>"Release of P-TEFb by flavopiridol treatment of cells causes a conformational change in 7SK snRNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:56:40"}
  • {"files"=>["https://ndownloader.figshare.com/files/835285"], "description"=>"<p>HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012335#s4\" target=\"_blank\">Materials and Methods</a>. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.</p>", "links"=>[], "tags"=>["p-tefb", "binding", "brd4", "causes", "7sk", "snrnp"], "article_id"=>505651, "categories"=>["Biochemistry", "Molecular Biology"], "users"=>["Brian J. Krueger", "Katayoun Varzavand", "Jeffrey J. Cooper", "David H. Price"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012335.g003", "stats"=>{"downloads"=>2, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_P_TEFb_binding_region_of_Brd4_causes_a_release_of_P_TEFb_from_the_7SK_snRNP_in_vivo_/505651", "title"=>"The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:56:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/835475"], "description"=>"<p>A) Western analysis of P-TEFb release from the 7SK snRNP after co-incubation of Tat. LARP7 and Cdk9 were analyzed by Western. I, Input; C, Control; T, Tat treated; Bound, bound to the beads; FT, flowthrough from the beads. B) and C) CMCT reactivity was determined as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012335#s4\" target=\"_blank\">Materials and Methods</a> using extension of the indicated primers. D) Western blot of the Tat treated 7SK snRNP probed for Cdk9 and HEXIM1. Indicated amounts of Tat were added to the 7SK snRNP. E) Graphical summary of the 7SK conformational change data. Bases in single stranded regions are boxed in black and bases in double stranded regions are unboxed. An asterisk denotes a change in CMCT reactivity and a minus sign indicates no change.</p>", "links"=>[], "tags"=>["hiv", "tat", "causes", "conformational", "7sk", "snrna", "hexim1"], "article_id"=>505842, "categories"=>["Biochemistry", "Molecular Biology"], "users"=>["Brian J. Krueger", "Katayoun Varzavand", "Jeffrey J. Cooper", "David H. Price"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0012335.g005", "stats"=>{"downloads"=>0, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_P_TEFb_release_by_HIV_Tat_causes_a_conformational_change_in_7SK_snRNA_and_release_of_HEXIM1_from_the_complex_/505842", "title"=>"P-TEFb release by HIV Tat causes a conformational change in 7SK snRNA and release of HEXIM1 from the complex.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:57:09"}

PMC Usage Stats | Further Information

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