The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain.
Heterologous expression of human LINE-1 ORF2 in yeast yielded a single polypeptide (Mr145 000) which reacted with specific antibodies and co-purified with a reverse transcriptase activity not present in the host cells. Various deletion derivatives of the ORF2 polypeptide were also synthesized. Reverse transcriptase assays using synthetic polynucleotides as template and primer revealed that ORF2 protein missing a significant portion of the N-terminal endonuclease domain still retains some activity. Deletion of the C-terminal cysteine-rich motif reduces activity only a small amount. Three non-overlapping deletions spanning 144 amino acids just N-terminal to the common polymerase domain of the ORF2 protein were analyzed for their effect on reverse transcriptase activity; this region contains the previously-noted conserved Z motif. The two deletions most proximal to the polymerase domain eliminate activity while the third, most-distal deletion had no effect. An inactive enzyme was also produced by substitution of two different amino acids in a highly-conserved octapeptide sequence, Z8, located within the region removed to make the deletion most proximal to the polymerase domain; substitution of a third had no effect. We conclude that the octapeptide sequence and neighboring amino acids in the Z region are essential for reverse transcriptase activity, while the endonuclease and cysteine-rich domains are not absolutely required.
Clements
A P
AP
Laboratory of Biochemistry, National Cancer Institute, Building 37, Room 4A-01, Bethesda, MD 20892, USA.
Singer
M F
MF
D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D016296:Mutagenesis; D016366:Open Reading Frames; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013379:Substrate Specificity; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C077214:L1Hs-encoded protein p40, human; D012194:RNA-Directed DNA Polymerase