Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme
The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled us...
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| Veröffentlicht in: | FEBS letters 1992-04, Vol.301 (1), p.23-28 |
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| creator | Dirani-Diab, Rim El Andreola, Marie-Line Nevinsky, Georgyi Tharaud, Danièle Barr, Philip J. Litvak, Simon Tarrago-Litvak, Laura |
| description | The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5′ modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT Forms. The effect of primer tRNA
Lys on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA
Lys, like RT p66/p66, when KCl was present in the incubation mixture. |
| doi_str_mv | 10.1016/0014-5793(92)80202-R |
| format | Article |
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Lys on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA
Lys, like RT p66/p66, when KCl was present in the incubation mixture.</description><identifier>ISSN: 0014-5793</identifier><identifier>EISSN: 1873-3468</identifier><identifier>DOI: 10.1016/0014-5793(92)80202-R</identifier><identifier>PMID: 1280600</identifier><identifier>CODEN: FEBLAL</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Affinity labeling ; Affinity Labels ; AIDS/HIV ; Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; DNA Replication ; DNA, Viral - biosynthesis ; Dose-Response Relationship, Drug ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; HIV Reverse Transcriptase ; HIV-1 ; HIV-1 - enzymology ; Oligodeoxyribonucleotides - metabolism ; p51 subunit ; Potassium Chloride - pharmacology ; Primer tRNA interaction ; Protein Conformation ; Recombinant form ; Recombinant Proteins - metabolism ; Reverse transcriptase ; RNA, Transfer, Lys - pharmacology ; RNA-Directed DNA Polymerase - chemistry ; RNA-Directed DNA Polymerase - drug effects ; RNA-Directed DNA Polymerase - genetics ; RNA-Directed DNA Polymerase - isolation & purification ; Transferases ; Yeasts - genetics</subject><ispartof>FEBS letters, 1992-04, Vol.301 (1), p.23-28</ispartof><rights>1992</rights><rights>FEBS Letters 301 (1992) 1873-3468 © 2015 Federation of European Biochemical Societies</rights><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c441R-5036cd69137ddad291fac20b8ec217057272f402188684fa52c9f2539ff82ae73</citedby><cites>FETCH-LOGICAL-c441R-5036cd69137ddad291fac20b8ec217057272f402188684fa52c9f2539ff82ae73</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0014-5793(92)80202-R$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,780,784,3548,27922,27923,45993</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5252669$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1280600$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Dirani-Diab, Rim El</creatorcontrib><creatorcontrib>Andreola, Marie-Line</creatorcontrib><creatorcontrib>Nevinsky, Georgyi</creatorcontrib><creatorcontrib>Tharaud, Danièle</creatorcontrib><creatorcontrib>Barr, Philip J.</creatorcontrib><creatorcontrib>Litvak, Simon</creatorcontrib><creatorcontrib>Tarrago-Litvak, Laura</creatorcontrib><title>Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme</title><title>FEBS letters</title><addtitle>FEBS Lett</addtitle><description>The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5′ modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT Forms. The effect of primer tRNA
Lys on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA
Lys, like RT p66/p66, when KCl was present in the incubation mixture.</description><subject>Affinity labeling</subject><subject>Affinity Labels</subject><subject>AIDS/HIV</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>DNA Replication</subject><subject>DNA, Viral - biosynthesis</subject><subject>Dose-Response Relationship, Drug</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>HIV Reverse Transcriptase</subject><subject>HIV-1</subject><subject>HIV-1 - enzymology</subject><subject>Oligodeoxyribonucleotides - metabolism</subject><subject>p51 subunit</subject><subject>Potassium Chloride - pharmacology</subject><subject>Primer tRNA interaction</subject><subject>Protein Conformation</subject><subject>Recombinant form</subject><subject>Recombinant Proteins - metabolism</subject><subject>Reverse transcriptase</subject><subject>RNA, Transfer, Lys - pharmacology</subject><subject>RNA-Directed DNA Polymerase - chemistry</subject><subject>RNA-Directed DNA Polymerase - drug effects</subject><subject>RNA-Directed DNA Polymerase - genetics</subject><subject>RNA-Directed DNA Polymerase - isolation & purification</subject><subject>Transferases</subject><subject>Yeasts - genetics</subject><issn>0014-5793</issn><issn>1873-3468</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkU2LFDEQhoMo67j6DxRyEFkPrfnoj_RlwV12VFgQBj2HTLpCRzrJmHSPzP4b_-mmp8f1pp5CVb1Vb6UehF5S8o4SWr8nhJZF1bT8omVvBWGEFZtHaEVFwwte1uIxWj1InqJnKX0nORa0PUNnlAlSE7JCv65s0D04q9WAda-i0iNEe6dGGzwOBo894F1FcZq2xeTtOOf6ySmPrXOTDx0Yqy14fcB7G6eEI-whJsBjVD7paHejypH1uA8uFFj5DveQPUJnXXbSuUEHt7Ve-RGbEF36bQv-7uDgOXpi1JDgxek9R9_WN1-vPxW3Xz5-vv5wW-iypJuiIrzWXd1S3nSd6lhLjdKMbAVoRhtSNaxhpiSMClGL0qiK6dawirfGCKag4efozTJ3F8OPCdIonU0ahkF5CFOSDeclIaL6p5DWVHBOSBaWi1DHkFIEI3fROhUPkhI5I5QzHznzkS2TR4Ryk9teneZPWwfdn6aFWa6_PtVVytRMvrO26UFWsYrVdZtl60X20w5w-C9rub65YnNhzrfsmJ33uVwGQb7-3kKU6QgcOpvRjbIL9u8fugf9MM3C</recordid><startdate>19920413</startdate><enddate>19920413</enddate><creator>Dirani-Diab, Rim El</creator><creator>Andreola, Marie-Line</creator><creator>Nevinsky, Georgyi</creator><creator>Tharaud, Danièle</creator><creator>Barr, Philip J.</creator><creator>Litvak, Simon</creator><creator>Tarrago-Litvak, Laura</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>6I.</scope><scope>AAFTH</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>7U9</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>19920413</creationdate><title>Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme</title><author>Dirani-Diab, Rim El ; Andreola, Marie-Line ; Nevinsky, Georgyi ; Tharaud, Danièle ; Barr, Philip J. ; Litvak, Simon ; Tarrago-Litvak, Laura</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c441R-5036cd69137ddad291fac20b8ec217057272f402188684fa52c9f2539ff82ae73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Affinity labeling</topic><topic>Affinity Labels</topic><topic>AIDS/HIV</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>DNA Replication</topic><topic>DNA, Viral - biosynthesis</topic><topic>Dose-Response Relationship, Drug</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>HIV Reverse Transcriptase</topic><topic>HIV-1</topic><topic>HIV-1 - enzymology</topic><topic>Oligodeoxyribonucleotides - metabolism</topic><topic>p51 subunit</topic><topic>Potassium Chloride - pharmacology</topic><topic>Primer tRNA interaction</topic><topic>Protein Conformation</topic><topic>Recombinant form</topic><topic>Recombinant Proteins - metabolism</topic><topic>Reverse transcriptase</topic><topic>RNA, Transfer, Lys - pharmacology</topic><topic>RNA-Directed DNA Polymerase - chemistry</topic><topic>RNA-Directed DNA Polymerase - drug effects</topic><topic>RNA-Directed DNA Polymerase - genetics</topic><topic>RNA-Directed DNA Polymerase - isolation & purification</topic><topic>Transferases</topic><topic>Yeasts - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dirani-Diab, Rim El</creatorcontrib><creatorcontrib>Andreola, Marie-Line</creatorcontrib><creatorcontrib>Nevinsky, Georgyi</creatorcontrib><creatorcontrib>Tharaud, Danièle</creatorcontrib><creatorcontrib>Barr, Philip J.</creatorcontrib><creatorcontrib>Litvak, Simon</creatorcontrib><creatorcontrib>Tarrago-Litvak, Laura</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>FEBS letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dirani-Diab, Rim El</au><au>Andreola, Marie-Line</au><au>Nevinsky, Georgyi</au><au>Tharaud, Danièle</au><au>Barr, Philip J.</au><au>Litvak, Simon</au><au>Tarrago-Litvak, Laura</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme</atitle><jtitle>FEBS letters</jtitle><addtitle>FEBS Lett</addtitle><date>1992-04-13</date><risdate>1992</risdate><volume>301</volume><issue>1</issue><spage>23</spage><epage>28</epage><pages>23-28</pages><issn>0014-5793</issn><eissn>1873-3468</eissn><coden>FEBLAL</coden><abstract>The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5′ modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT Forms. The effect of primer tRNA
Lys on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA
Lys, like RT p66/p66, when KCl was present in the incubation mixture.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>1280600</pmid><doi>10.1016/0014-5793(92)80202-R</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | FEBS letters, 1992-04, Vol.301 (1), p.23-28 |
| issn | 0014-5793 1873-3468 |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present); EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | Affinity labeling Affinity Labels AIDS/HIV Analytical, structural and metabolic biochemistry Biological and medical sciences DNA Replication DNA, Viral - biosynthesis Dose-Response Relationship, Drug Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology HIV Reverse Transcriptase HIV-1 HIV-1 - enzymology Oligodeoxyribonucleotides - metabolism p51 subunit Potassium Chloride - pharmacology Primer tRNA interaction Protein Conformation Recombinant form Recombinant Proteins - metabolism Reverse transcriptase RNA, Transfer, Lys - pharmacology RNA-Directed DNA Polymerase - chemistry RNA-Directed DNA Polymerase - drug effects RNA-Directed DNA Polymerase - genetics RNA-Directed DNA Polymerase - isolation & purification Transferases Yeasts - genetics |
| title | Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme |
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