Figure 6

Analysis of the RNA-binding capacity of the rLbRPA-1 protein and its relative affinity for RNA and ssDNA. (Panel A) Two hundred ng of digoxigenin labeled 3′ HSP70 UTR-II were incubated with beads containing different amounts of rLbRPA-1: 0 (dot 1), 0.05 pM (2), 0.25 pM (3), 1.25 pM (4), 6.25 pM (5) and 31.25 pM (6). After whasing out of the unbound RNA, eluted complexes were deposited on the membrane and the remaining bound RNA was monitored by an anti-digoxigenin antibody. (Panel B) One pM of either denatured (dot D) or renatured rLbRPA-1 (dot R) was incubated with the digoxigenin-labeled RNA oligonucleotide (100 fM). Additionally, the binding of the Dig-RNA oligonucleotide to the renatured rLbRPA-1 was competited with increasing amounts of either the non-labelled RNA oligonucleotide (upper array) or a non-labelled DNA oligonucleotide (lower array) containing the equivalent sequence: 25 fM (dot 1), 50 fM (2), 100 fM (3) and 200 fM (4). After whasing out the unbound oligonucleotides, the complexes were eluted and put on the membrane, which was revealed by an anti-digoxigenine antibody. (Panel C) The membrane of panel B was incubated with an anti-his-tag antibody in order to monitor that equivalent amounts of rLbRPA-1 was present in the different samples.