TRM1 binds specifically to the HOMO region of the +723 to +983 DNA
sequence of rbcS-m3. Gel mobility-shift assays were as
described in Materials and Methods. (A)
Binding to wild-type DNA. Lane 1, probe +723 to +983 DNA only
(Control); lane 2, probe DNA plus TRM1; lanes 3–5, as in lane 2 but
with competing wild-type DNA added; lanes 6–8, as in lane 2 but with
mutated (M) DNA added; lanes 9–11, as in lane 2 but with nonspecific
(NS) DNA added. Lanes 3, 6, and 9 contain 3.4 ng of competitor DNA;
lanes 4, 7, and 10 contain 17 ng of competitor DNA; lanes 5, 8, and 11
contain 85 ng of competitor DNA. The wild-type competitor was unlabeled
+723 to +983 bp DNA (W). The mutant (M) competitor was +723 to +983 bp
DNA with HOMO mutations (see Materials and Methods) in
the TRM1 binding site. A 100-bp DNA ladder (NS) (GIBCO/BRL) was used
as the nonspecific (NS) competitor DNA. (B) TRM1 binds
to wild-type rbcS-m3 +723 to +983 DNA but not to this
sequence with mutations in the HOMO region. Lane 1, TRM1 plus wild-type
probe DNA, as in A, lane 2 above; lane 2, TRM1 protein
plus labeled HOMO mutant DNA.