Interference footprinting analysis of telomerase elongation complexes.
Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single (32)P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3'-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3'-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.
Benjamin
S
S
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Baran
N
N
Manor
H
H
D000818:Animals; D001665:Binding Sites; D018983:DNA Footprinting; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D019098:Telomerase / Q000235:genetics*; D013698:Templates, Genetic; D013768:Tetrahymena
D017931:DNA Primers; D004277:DNA, Single-Stranded; D019098:Telomerase