Journal: Frontiers in Plant Science
Article Title: The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii
Figure Lengend Snippet: RNA structure analyses of the psbC 5′ UTR by enzymatic probing. In vitro transcribed RNA corresponding to the wild-type and mutant psbC 5′ UTR were untreated (0) or digested with RNase T1 (T1) or RNase V1 (V1), which cleave unpaired G residues or RNA helices, respectively. Digestion products were resolved by denaturing PAGE and revealed by autoradiography. The positions of cleavage sites are indicated with respect to their positions on the 5′ UTR, based on the motilities of molecular size markers (not shown). (A) The substrate RNA was 3′- 32 P-labeled wild-type psbC 5′ UTR. (B,C) The substrates were 5′- 32 P-end-labeled RNAs corresponding to the psb C 5′ UTR with wild-type sequence (wt), or carrying one of the mutations; psbC-FuD34 (m), or psbC-F34suI (s). The exact position of certain digestion products could not be determined with certainty ( ∗ ).
Article Snippet: Digestions with RNase T1 (Fermentas) and RNase V1 (Ambion) were performed with approximately 3 × 105 cpm RNA in 10 μl 10 mM Tris [pH 7], 100 mM KCl, 10 mM MgCl2 [Ambion] and either 1μg sheared yeast RNA (10 mg/ml, Ambion) and 1 mU of RNase V1 (1 U/μl, Ambion) for 5 min at 24°C, or 4 μg sheared yeast RNA (Sigma Chemicals) and 500 mU of RNase T1 (100 U/μl, Fermentas) for 2 min at 24°C.
Techniques: In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography, Labeling, Sequencing