title: HIV-1 Reverse Transcriptase Polymerase and RNase H (Ribonuclease H) Active Sites Work Simultaneously and Independently.
abstract: HIV reverse transcriptase plays a central role in viral replication and requires coordination of both polymerase and RNase H activities. Although this coordination is crucial in viral replication, whether a DNA/RNA hybrid can simultaneously engage both active sites has yet to be determined as structural and kinetic analyses have provided contradictory results. Single nucleotide incorporation and RNase H cleavage were examined using presteady-state kinetics with global data analysis. The results revealed three interconverting reverse transcriptase-DNA/RNA species; 43% were active for both sites simultaneously, 27% showed only polymerase activity, and the remaining 30% were nonproductive. Our data clearly demonstrated that the DNA/RNA hybrid could contact both active sites simultaneously, although the single nucleotide incorporation (105 s-1) was ∼5-fold faster than the cleavage (23 s-1). By using a series of primers with different lengths, we found that a string of at least 4-6 nucleotides downstream of the cleaving site was required for efficient RNA cleavage. This was corroborated by our observations that during processive nucleotide incorporation, sequential rounds of RNA cleavage occurred each time after ∼6 nucleotides were incorporated. More importantly, during processive primer extension, pyrophosphate (PPi) release was rate-limiting so that the average rate of nucleotide incorporation (∼28 s-1) was comparable with that of net RNA cleavage (∼27 nucleotides(s)). Although polymerization is efficient and processive, RNase H is inefficient and periodic. This combination allows the two catalytic centers of HIVRT to work simultaneously at similar speeds without being tightly coupled.
authors: From the The University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.
authors: An
authors: A
authors: Li
authors: From the The University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712.
authors: Jiawen
authors: J
authors: Li
authors: From the The University of Texas at Austin, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, Austin, Texas 78712 kajohnson@mail.utexas.edu.
authors: Kenneth A
authors: KA
authors: Johnson
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D015497:HIV-1 / Q000201:enzymology*; D012313:RNA / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry*
chemical_list: D012313:RNA; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H
keywords: DNA polymerase; RNase H; cooperativity; enzyme kinetics; global data fitting; human immunodeficiency virus (HIV); reverse transcriptase; reverse transcription; transient kinetics