Mechanistic studies of early pausing events during initiation of HIV-1 reverse transcription.
We have investigated the role of sequences that surround the primer binding site (PBS) in the reverse transcriptase-mediated initiation of (-) strand DNA synthesis in human immunodeficiency virus type 1. In comparisons of reverse transcription initiated from either the cognate primer tRNALys.3 or a DNA primer D-Lys.3, bound to PBS sequences, we observed that a +3 pausing site occurred in both circumstances. However, the initiation reaction with tRNALys.3 was also characterized by a pausing event after incorporation of the first nucleotide. Alteration of sequences at the 5'-end instead of the 3'-end of the PBS resulted in elimination of the +3 pausing site, suggesting that this site was template sequence-dependent. In contrast, the pausing event at the +1 nucleotide position was still present in experiments that employed either of these mutated RNA templates. The mutations at the 5'-end of the PBS also caused a severely diminished rate of initiation and the strong arrest of reactions at the +1 stage when tRNALys.3 was used as primer. Therefore, we propose that the +1 pausing event is an initiation-specific event in regard to reactions primed by tRNALys.3 and that sequences at the 5'-end of the PBS may facilitate the release of reverse transcription from initiation to elongation.
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada.
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Liang
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Rong
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Götte
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Li
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Quan
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Kleiman
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Wainberg
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase