Figure 5.

Differential effects of UV irradiation on expression of Sulfolobus tfb genes. (a) Both S. solfataricus and S. acidocaldarius encode three homologues of the basal transcription factor TFB. TFB1 and TFB2 are full-length orthologues, but TFB3 is severely truncated, lacking the B-finger domain (B) for transcription initiation and the HTH domain for binding to the BRE. C's represent the cysteines implicated in zinc binding (b) In both S. solfataricus and S. acidocaldarius, UV irradiation causes a dramatic increase in transcription of tfb3 and a significant reduction in expression of tfb2, whilst tfb1 levels are unaffected. Ratios of gene expression for the UV treated and control cultures are expressed in a log base 2 format, so that a value of 1 represents a two-fold increase in transcription. S. acidocaldarius and S. solfataricus data points are represented by triangles and circles, respectively. (c) Immunodetection of endogenous RNA polymerase (RNAP), TFB1 and TFB3 from cell lysates from control and UV-treated cells. Cells were harvested and lysed after 90 minutes of treatment. Levels of TFB3 protein increase after UV treatment in agreement with microarray data and quantitative RT-PCR (Table 2). (d) SDS-PAGE and western blot of immunoprecipitation of RNAP using specific antibodies (B' subunit) and co-immunoprecipitation of bound TFB1 and TFB3. Increasing concentrations of TFB3 (0-480 nM) were added to a mix with constant concentrations of RNAP and TFB1 (40 nM:80 nM). In the control lane where RNAP was not added, concentrations of TFB1 and TFB3 were 80 nM and 480 nM, respectively. The molar ratios of TFB3 and TFB1 are indicated below the TFB3 concentrations. Relative amounts of TFB1 bound to RNAP in each sample are indicated, showing a decrease of TFB1 bound to RNAP as TFB3 increases. All values are normalized with the total amount of RNAP immunoprecipitated in each sample.