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

摘要: HIV reverse transcriptase plays a central role in viral replication and requires coordination of both polymerase RNase H activities. Although this is crucial replication, whether DNA/RNA hybrid can simultaneously engage active sites has yet to be determined as structural kinetic analyses have provided contradictory results. Single nucleotide incorporation cleavage were examined using presteady-state kinetics with global data analysis. The results revealed three interconverting transcriptase-DNA/RNA species; 43% for simultaneously, 27% showed only activity, the remaining 30% nonproductive. Our clearly demonstrated that could contact although single (105 s−1) was ∼5-fold faster than (23 s−1). By series primers different lengths, we found string at least 4–6 nucleotides downstream cleaving site required efficient RNA cleavage. This corroborated by our observations during processive incorporation, sequential rounds occurred each time after ∼6 incorporated. More importantly, primer extension, pyrophosphate (PPi) release rate-limiting so average rate (∼28 comparable net (∼27 nucleotides(s)). polymerization processive, inefficient periodic. combination allows two catalytic centers HIVRT work similar speeds without being tightly coupled.