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 (M-r 145 000) which reacted with specific antibodies and co-purified with a reverse transcriptase activity not present in the h ost cells. Various deletion derivatives of the ORF2 polypeptide were a lso synthesized. Reverse transcriptase assays using synthetic polynucl eotides as template and primer revealed that ORF2 protein missing a si gnificant 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 th e 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 a ctivity while the third, most-distal deletion had no effect. An inacti ve enzyme was also produced by substitution of two different amino aci ds in a highly-conserved octapeptide sequence, Z(8), located within th e region removed to make the deletion most proximal to the polymerase domain; substitution of a third had no effect. We conclude that the oc tapeptide sequence and neighboring amino acids in the Z region are ess ential for reverse transcriptase activity, while the endonuclease and cysteine-rich domains are not absolutely required.