HIV-1 Reverse Transcriptase Polymerase and RNase H (Ribonuclease H) Active Sites Work Simultaneously and Independently.

An Li, Jiawen Li, Kenneth A Johnson

The Journal of biological chemistry 2016 Dec 16

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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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Citation

An Li, Jiawen Li, Kenneth A Johnson. HIV-1 Reverse Transcriptase Polymerase and RNase H (Ribonuclease H) Active Sites Work Simultaneously and Independently. The Journal of biological chemistry. 2016 Dec 16;291(51):26566-26585