Fig. 3.

Determination of the nupN transcription start point and CodY-binding regions. (A) Primer extension analysis of the nupN mRNA. Primer oBB102 annealing to the lacZ gene of the nupN276-lacZ fusion was extended with reverse transcriptase using as the template total RNA from fusion-containing strains BB2809 (wt) and BB2819 (codY) grown in the 16-amino-acid-containing medium. The sequence of the template strand of pBB1520 determined from reactions primed with oBB102 is shown to the left. The apparent transcription start site of the nupN gene is in bold and marked by the +1 notation. A bent arrow indicates the direction of transcription. (B) DNase I footprinting analysis of CodY binding to the nupN regulatory region. The nupNp⁺ DNA fragments labeled on the template strand were incubated with increasing amounts of purified CodY in the presence of 10 mM ILV and 2 mM GTP and then with DNase I. The apparent transcription start site and direction of nupN transcription are shown by the bent arrow. The protected areas are indicated by the vertical lines. CodY concentrations used (nanomolar concentrations of monomers) are indicated above each lane. (C) Same as panel B, nupN120p⁺ fragment.