Figure 4. Temperature-dependent structural alterations of avashort-RNAT. (A) Secondary structure prediction of the avashort-5′UTR computed with mfold,86 including 72 Nt coding region (cdr) as well as the first three Nt of the blunt end restriction site EcoRV. SD and AUG are highlighted by gray circles. Black encircled Nt are referred to in the quantification in (C). The 5′ structure forms three hairpins (H1-H3), the SD sequence is partly base paired in H2, and the AUG is embedded in H3. The cdr forms H4 and H5. (B) In vitro transcribed avashort RNA (avashort-5′UTR and 72 Nt) was 5′ labeled and partially digested with RNase T1 (0.001U) and RNase V1 (0.0025U) at the indicated temperatures. For orientation, an alkaline ladder (LOH) was loaded; for the control (C) enzyme was replaced by A. dest. (C) Quantification of altered RNase T1 cleavage intensity between 25–42 °C for selected guanines. Band intensities were quantified from (B) using the Alpha Ease software (Alpha Innotech, Biozym). Shown are relative intensities for each Nt position with band intensity at 30 °C set as 1.