The Crystal Structure of the Monomeric Reverse Transcriptase from Moloney Murine Leukemia Virus

Abstract

Reverse transcriptases (RTs) are multidomain enzymes of variable architecture that couple both RNA- and DNA-directed DNA polymerase activities with an RNase H activity specific for an RNA:DNA hybrid in order to replicate the single-stranded RNA genome of the retrovirus. Previous structural work has been reported for the heterodimeric HIV-1 and HIV-2 RTs. We now report the first crystal structure of the full-length Moloney murine leukemia virus (MMLV) RT at 3.0 A resolution. The structure reveals a clamp-shaped molecule resulting from the relative positions of the thumb, connection, and RNase H domains that is strikingly different from the HIV-1 RT and provides the first example of a monomeric reverse transcriptase. A comparative analysis with related DNA polymerases suggests a unique trajectory for the template-primer exiting the polymerase active site and provides insights regarding processive DNA synthesis by MMLV RT.

Insight into the Mechanism of the Stabilization of Moloney Murine Leukaemia Virus Reverse Transcriptase by Eliminating RNase H Activity

“…MMLV RT and the and subunits of AMV RT comprise the fingers, palm, thumb, connection, and RNase H domains. 5,6) The active site of DNA polymerase reaction resides in the fingers/palm/thumb domain, while that of RNase H reaction lies in the RNase H domain.…”

Gln84 of moloney murine leukemia virus reverse transcriptase regulates the incorporation rates of ribonucleotides and deoxyribonucleotides

“…Whether the mutation affects the enzyme's other properties remains to be determined. Nonetheless, since the structure of MMLV-RT is significantly different from the structure of HIV-RT [4,17], it would not be surprising E/P-T Fig. 5.…”

“…Based on the analysis of the crystal structure of MMLV-RT ( [3,4] and Fig. 1), we inferred that Q84 might be a residue involved in the enzyme's interaction with the template-primer and speculated that substitution of this bulky residue with smaller ones might improve the enzyme's ability to synthesize RNA.…”

“…Through the RNAdependent and DNA-dependent DNA polymerase activities and the RNase H activity, RT converts the single-stranded viral RNA genome into a double-stranded integration-competent DNA during reverse transcription [2]. X-ray crystallographic structure studies revealed that the polymerase domain of MMLV-RT, like other nucleic acid polymerases, is shaped like a half-opened right hand, comprising of the palm, fingers and thumb subdomains [3,4]. The RNase H domain is connected to the polymerase domain through the ''connection'' domain and positioned close to the thumb subdomain.…”

Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

“…1). 13) On the assumption that MMLV RT stability increases upon introduction of the charged residue into one of these consecutive surface hydrophobic residues, 10 variants (F303R, F303K, L304R, L304K, L432R, L432K, V433R, V433K, I434R, and I434K) were *Corresponding author. Email: yasukawa@kais.kyoto-u.ac.jp Bioscience, Biotechnology, and Biochemistry, 2014 Vol.…”