title: Mn2+ suppressor mutations and biochemical communication between Ty1 reverse transcriptase and RNase H domains.
abstract: Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn(2+) inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn(2+) inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn(2+) inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1 Delta cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1(+) cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn(2+) inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.
authors.affiliation: Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 733 N. Broadway, Baltimore MD 21205, USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Yarrington
authors.forename: Jichao
authors.initials: J
authors.lastname: Chen
authors.forename: Eric C
authors.initials: EC
authors.lastname: Bolton
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D000595:Amino Acid Sequence; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology; D008345:Manganese / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018626:Retroelements; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013489:Suppression, Genetic
chemical_list: D004791:Enzyme Inhibitors; D018626:Retroelements; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H