Characterization of Active Reverse Transcriptase and Nucleoprotein Complexes of the Yeast Retrotransposon Ty3 in Vitro
Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. I...
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Published in | The Journal of biological chemistry Vol. 274; no. 51; pp. 36643 - 36648 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
17.12.1999
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Subjects | |
Online Access | Get full text |
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Summary: | Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and
a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1
replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5â² RNA and primer tRNA 3
Lys catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions
in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes
and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes
annealing of primer tRNA i
Met to a 5â²-3â² bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5â² PBS ( 1 ). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3
RNA with the 5â²-3â² PBS, primer tRNA i
Met , NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine
leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs,
retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes
were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA
dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3
NCp9 is an ancestor of HIV-1 NCp7. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.274.51.36643 |