Figure 2
The 5′ 10-bp sequence adjoining the HSE controls the activity of the cryab promoter. (A) Cryab promoter sequences (−896/+1), showing the dimeric HSE (−392/−383) and sequences from −64 to −23 that includes the trimeric HSE (numbers 1–6). The thin arrows on top of the trimeric HSE (blue) indicate the 5'-NGAAN-3' inverted orientation of the HSE motifs. Mutations are indicated in red and underlined (numbers 2–6, 8, 9). The complete promoter is −896 to +44, and the truncated promoter includes sequences −64 to +44. tGFP (green) starts with ATG. TSS is shown with an arrow at +1. The dimeric HSE (−392/−383) and sequences at −64/−35 (HSE-αB) and −29/−23 (TATA box) are schematically represented with open boxes in the promoter/reporter constructs. The corresponding activity of each construct (relative tGFP expression as assessed by RT-qPCR) is shown on the right (means ± standard deviation). Note inhibition of tGFP expression (numbers 5, 6, 9) by mutations in the 10-bp sequence (−64/−55) adjoining the HSE. The numbers (1 − 9) in the top panel with relevant sequences correspond to numbers for promoter/reporter constructions in the lower panel. Note that deletion of the dimeric HSE at −392/−383 does not affect the reporter expression in these cells (1 and 2). (B) Sequences on the 3' end of the trimeric HSE do not impact the expression significantly. Relevant sequences are shown on top. Mutations are indicated in red and are underlined. Schematics of various constructions and the activity of each promoter/reporter construct, as assessed by RT-qPCR (means ± standard deviation), is shown as in A. The key to various notations in A and B is given on the bottom.