The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-reader (also called the B-finger) motif. TFB2 functions, though poorly, in promoter-dependent transcription initiation. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening. Archaeal RNA polymerase requires two transcription factors for initiation: TBP, which binds to TATA boxes, and TFB, which binds TBP and DNA, recruits RNAP and helps initiate transcription. Archaeal TFBs usually contain a conserved B-reader sequence homologous to the eukaryotic B-reader motif in their N-terminal domains. This region is involved in the assembly of the transcription complex, promoter melting and in transcription start site determination but its position and orientation relative to promoter DNA during initiation is not clear. In this study the positioning of the TFB B-reader relative to DNA was determined by cross-linking using TFB variants substituted with photoactivatable unnatural amino acids. The results demonstrate that the B-reader is in close proximity to the transcription start site on the template but not the non-template strand in transcription initiation complexes. Furthermore, the position of the B-reader varies between closed and open promoter complexes, and between open promoter and early initiation complexes. Thus the archaeal B-reader sequence is poised to interact with promoter DNA in a dynamic fashion, and is likely playing a role in positioning the template-strand in an open pre-initiation complex.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-1277 |
| Date | 01 January 2010 |
| Creators | Micorescu, Michael |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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