Figure 3.

Determination of T. brucei telomerase template by direct synthesis. (A) Semipurified DEAE fraction was used as telomerase source to elongate permuted telomeric primers (see Table 1 for primer sequences and names). The most intense bands correspond to the putative template translocation/pause position. Duplicated reactions were performed in which extracts were or were not RNase A-pretreated as indicated (+ or −). Lane M is oligonucleotide tel 6 labeled with terminal transferase and [α-³²P]dCTP. (B) Two different oligonucleotide (tel 6, lane 1 and tel 1, lane 2) were 3′ end labeled with terminal transferase and [α-³²P]dGTP to demonstrate that primers labeled with dGTP more accurately indicate the primer +1 nucleotide position (19 on the left side of the gel). Lanes 3 and 5 show, respectively, standard telomerase reactions using oligonucleotides tel 1 and tel 2 as enzyme substrates. (C) Hypothetical alignment of the permuted primers in the putative T. brucei telomerase RNA template region. Capital letters represent the sequence of the input primers and small letters indicate nucleotides added by a single round of telomerase polymerization, according to the results obtained in A and B. The number of residues added to the 3′ end of each primer to reach the translocation/pause position is indicated in parentheses according to the results obtained in A and B.