The upstream region of known MsvR-encoding genes contains at least two of these binding boxes, suggesting that these boxes may serve as DNA recognition sequences for auto-regulation by the MsvR family proteins. The binding boxes for MthMsvR overlap the transcription start site in Mth P fpaA and the BRE/TATA box in Mth P msvR . MthMsvR binding to box(es) GSK458 mw two and three have been shown to prevent binding of TBP and TFB to Mth P msvR [9], suggesting that MthMsvR acts as a transcription repressor. Ma P msvR contains two MsvR binding boxes, A and B, corresponding

to Mth P msvR/fpaA boxes 2 and 3, respectively (Figure 1b) [9]. In contrast to the seventy-three-nucleotide 5′ untranslated region (UTR) in the Mth msvR transcript [9], transcription start site mapping of the Ma msvR transcript indicates that transcription initiates at a G nucleotide eight nucleotides upstream of the ATG start codon (Figure 1c).

The shorter 5′ UTR of Ma msvR is consistent with the results of transcription start site mapping in the closely related Methanosarcina mazei Gö1, where the msvR (MM2525) transcript was classified as leaderless for having a 5′ UTR of less than ten nucleotides [21]. A TATA box is centered 27 nucleotides upstream of the Ma msvR transcription start site and boxes A and B are located upstream of the TATA box (Figure 1c). MaMsvR binding to box B likely blocks the purine-rich BRE element just upstream of the Ralimetinib molecular weight Ma P msvR TATA box, resulting in repression of transcription [9, 10, 22, 23]. Despite some differences in the placement of the MsvR binding boxes, it is likely that MsvR proteins repress transcription of their Tyrosine-protein kinase BLK own genes by blocking LDK378 research buy access to the promoter region. DNA binding behavior of MaMsvR varies under non-reducing and reducing conditions Electrophoretic mobility shift assays (EMSAs) were used to compare the binding of MaMsvR to Ma P msvR and Mth P msvR/fpaA

under non-reducing (+) and reducing (R) conditions (Figure 2a). Additionally, MthMsvR was tested for binding to Ma P msvR and MthMsvR binding to Mth P msvR/fpaA served as a control (Figure 2b). Both MaMsvR and MthMsvR bound to Ma P msvR and Mth P msvR/fpaA. However, MaMsvR bound only under reducing conditions, while MthMsvR bound both promoters under non-reducing and reducing conditions (Figure 2a, b). This was consistent with previously published results showing that MthMsvR bound Mth P msvR/fpaA under oxidizing and reducing conditions [9]. Neither protein showed notable binding to the well-described Mth histone control promoter (P hmtB ), which demonstrated the specificity of MsvR binding (Figure 2a,b) [24, 25]. Figure 2 EMSA of MsvR homologues on their respective promoters. The gel wells are indicated (W).