Fig. 3.

Functional validation and characterization of acorn worm TR. (A) Direct telomerase activity assay of acorn worm telomerase reconstituted in vitro. (top) Template sequence of acorn worm TR (open box) with base-pairing of six permuted telomeric DNA primers a–f. Sequence and number of expected nucleotides added are depicted for each primer. (bottom) Direct primer-extension assay of acorn worm telomerase. Acorn worm telomerase was in vitro reconstituted from T7 transcribed SkoTR (436 nt) and SkoTERT synthesized in RRL. The reconstituted acorn worm telomerase was analyzed with six permuted telomeric DNA primers (lanes 2–7). A reaction omitting SkoTR was included as a negative control (lane 1). A ³²P end-labeled 18-mer oligonucleotide was added to each reaction as recovery control (r.c.) prior to ethanol precipitation of DNA products. Numbers to the right of the gel indicate the number of repeats or nucleotides added to the primer. (B) Two essential fragments of SkoTR. The T-PK and CR4/5 fragments of SkoTR were synthesized separately and assembled with SkoTERT in RRL, followed by telomerase activity assay. The schematic secondary structures of the SkoTR T-PK (top) and CR4/5 (bottom) fragments. Nucleotide numbers denote the 5′- and 3′-ends of the T7 transcribed RNA fragments, T-PK and CR4/5. Positions of two highly conserved U residues (U289 and U290) in the P6.1 loop of CR4/5 domain are shown. (C) Minimal requirement of TR domains for telomerase activity. T7 transcribed SkoTR fragments, T-PK (nt 1–184) and CR4/5 (nt 237–307), were assembled with in vitro synthesized SkoTERT and analyzed for activity. The CR4/5 fragments with a P6.1 substitution (U289C or U290C) were assembled with T-PK fragment and SkoTERT and assayed for activity with the primer (GGGTTA)₃. A ³²P end-labeled 18-mer oligonucleotide was added to each reaction prior to ethanol precipitation of DNA products as r.c. The numbers of repeats added to the primer are shown to the right of the gel.