Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39
- University College London, United Kingdom
- Max Planck Institute for Terrestrial Microbiology, Germany
- University of Freiburg, Germany
Figures
Figure 1 with 2 supplements
Identification of a dimeric TFE factor in S. solfataricus.
(A) Conserved and acquired domains of TFIIEα and TFIIEβ-related proteins in Eukaryotes and Archaea derived from (Blombach et al., 2009; Vannini and Cramer, 2012). (B) Homologous expression and …
Figure 1—figure supplement 1
Sso0944 and TFEα form a dimeric complex.
Sso0944 was isolated as C-terminal His10-tag fusion from S. solfataricus M16 transformed with pMJ0503-Sso0944 under more stringent conditions. The salt concentration during the Ni-affinity …
Figure 1—figure supplement 2
Quantitative immunodetection of TFEα, TFEβ and TBP in S. solfataricus P2 cell lysates during exponential growth phase (n = 3).
18 µg of cell lysate (total soluble protein content) was loaded into each lane. Total protein-staining of the Western blots was performed using Ponceau S to verify loading of equal protein amounts. …
Figure 2 with 2 supplements
The C-terminal domain of Sso TFEβ harbours a 4Fe-4S cluster that is conserved in human RNAPIII subcomplex hRPC62/39.
(A) UV-vis spectrum of TFEα/β-His. (B) Cw-EPR spectra of TFEα/β prepared in the presence of 1 mM DTT with or without the addition of 20 mM Na-dithionite. Besides the main [3Fe-4S]+ cluster signal at …
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Figure 2—source data 1
Theoretical and experimentally calculated masses of proteins and protein complexes.
- https://doi.org/10.7554/eLife.08378.009
Figure 2—figure supplement 1
Temperature dependence of the cw-EPR spectra of Sso TFEαβ and hRPC62/39.
The decrease in signal at temperatures above 20 K is characteristic of cubane-like FeS clusters.
Figure 2—figure supplement 2
Nano-electrospray ionization mass spectrum of human hRPC62/C39 (top).
The +22 charge state, corresponding to the hRPC62/C39 + [4Fe-4S] complex was subjected to collision induced dissociation. At the low m/z region of the tandem mass spectrum, two charge state series …
Figure 3 with 1 supplement
Characterisation of the TFEα/β heterodimerization interface.
(A) Amino acid sequences and secondary structure of TFEα and TFEβ. The α-helices in the TFEα WH domain are numbered according to (Meinhart et al., 2003). Deletion (red boxes) and substitution …
Figure 3—figure supplement 1
Additional mutants of the N-terminal tip of α-helix 3 of TFEα and their effect on dimerization with TFEβ
Ni-affinity co-purification assays conducted for TFEβ-His co-expressed with TFEα (WT), TFEα K46E, TFEα D49T, or TFEα R51E/K52E. Red and blue triangles indicate the position of the respective mutant …
Figure 4
TFEαβ dimerization depends on the integrity of the Fe-S cluster.
Ni-affinity chromatography of TFEβ-His (WT), TFEβ-His C92S, TFEβ-His C95S, TFEβ-His C101S or TFEβ-His C112S co-expressed with TFEα. The graphs show the absorption profile monitored at 260 nm, 280 …
Figure 5
TFEα/β forms a stable interaction with the RNAP clamp module.
10 μM TFEα/β-His were incubated together with 10 μM recombinant RNAP clamp and the sample was resolved gel filtration. The presence of RNAP clamp and TFEα/β in the fractions was determined by …
Figure 6 with 1 supplement
TFEα/β increases the stability of the preinitiation complex (PIC) and promotes DNA melting.
(A) NTS sequence of the DNA templates for the promoters tested in electrophoretic mobility shift assay (EMSA) and potassium permanganate footprinting assays. The templates incude a 4 nt heteroduplex …
Figure 6—figure supplement 1
TFEα/β is part of the PIC.
Supershift EMSA with PICs formed in presence or absence of TFEα/β (0.1 µM) being exposed to antisera directed against TFEα, TFEβ and TBP. Antibody-PIC complexes migrate slower in the gel compared to …
Figure 7 with 2 supplements
TFEα/β stimulates abortive and productive transcription.
(A) Abortive transcription assays measuring ApGpA trinucleotide synthesis on −4/−1 heteroduplex and homoduplex SSV1-T6 promoter templates in the presence or absence of 1 µM TFEα/β. (B) NTS sequence …
Figure 7—figure supplement 1
Productive transcription assays using the SSB and Rpo5 promoters.
Circular relaxed plasmids with different S. solfataricus promoters fused to C-less cassettes were used as templates. The position of the run-off transcripts (asterisk) and its expected size is …
Figure 7—figure supplement 2
Effect of TFEα/β mutant variants on productive transcription on the Rpo5 promoter.
Transcription assays and quantification were carried out as described above with 3 µM TFEα/β or variant. The mean of three technical replicates is shown. Error bars depict 1× standard deviation.
Figure 8
Structure and evolution of TFEα/β.
(A) Model of the dimerization interface of TFEα/β and its interaction with the RNAP clamp based on data presented here and elsewhere (Grohmann et al., 2011; Grünberg et al., 2012; He et al., 2013). …
Tables
Table 1
Genetic experiments showing that the TFEβ encoding gene Saci_1342 is essential
| Parental strain | Plasmid integration relative to Saci_1342 | Number of clones tested | Clones with Saci_1342 deletion obtained |
|---|---|---|---|
| MW001 | upstream | 40 | 0 |
| downstream | 40 | 0 | |
| MW001 Saci_1162::Saci_1342 | upstream | 10 | 4 |
| downstream | 10 | 0 |
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Table 1—source data 1
Genetic experiments showing that the TFEβ encoding gene Saci_1342 is essential
- https://doi.org/10.7554/eLife.08378.007
Additional files
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Supplementary file 1
List of plasmids generated by restriction enzyme-based cloning.
- https://doi.org/10.7554/eLife.08378.022
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Supplementary file 2
List of plasmids generated by PCR-based Site-directed mutagenesis.
- https://doi.org/10.7554/eLife.08378.023
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Supplementary file 3
List of oligonucleotides used in this study.
- https://doi.org/10.7554/eLife.08378.024
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Supplementary file 4
List of in vitro transcription templates with promoters fused to a C-less cassette.
- https://doi.org/10.7554/eLife.08378.025
