Interference footprinting analysis of telomerase elongation complexes

Telomerase is a reverse transcriptase that adds single-stranded telomeric r epeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing revers e transcriptase motifs, and auxiliary proteins. We have carried out an inte rference footprinting analysis of the Tetrahymena telomerase elongation com plexes. In this study, single-stranded oligonucleotide primers containing t elomeric sequences were modified with base-specific chemical reagents and e xtended with the telomerase by a single P-32-labeled dGMP or dTMP. Base mod ifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the te lomerase and the six or seven 3'-terminal residues of the primers. These in teractions occurred not only with the RNA template region, but also with an other region in the enzyme ribonucleoprotein complex designated the telomer ase DNA interacting surface (TDIS), This was indicated by footprints genera ted with dimethyl sulfate (that did not affect Watson-Crick hydrogen bondin g) and by footprinting assays performed with mutant primers. In primers ali gned at a distance of 2 nucleotides along the RNA template region, the foot prints of the sis or seven 3'-terminal residues were shifted by 2 nucleotid es. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. W eak interactions occurred between the telomerase and residues located upstr eam of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.