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Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39.

Blombach F, Salvadori E, Fouqueau T, Yan J, Reimann J, Sheppard C, Smollett KL, Albers SV, Kay CW, Thalassinos K, Werner F - Elife (2015)

Bottom Line: The eukaryotic transcription factor TFIIE consists of α and β subunits.Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39.These activities are strictly dependent on the β subunit and the promoter sequence.

View Article: PubMed Central - PubMed

Affiliation: Institute for Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.

ABSTRACT
Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.

No MeSH data available.


Related in: MedlinePlus

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 SDS-PAGE and silver staining. The position of peaks for gel filtration marker proteins is indicated on top. For experiments with TFEα/β ΔWH the contrast was enhanced for the lower part of the gel in order to visualize TFEβ ΔWH.DOI:http://dx.doi.org/10.7554/eLife.08378.015
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fig5: 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 SDS-PAGE and silver staining. The position of peaks for gel filtration marker proteins is indicated on top. For experiments with TFEα/β ΔWH the contrast was enhanced for the lower part of the gel in order to visualize TFEβ ΔWH.DOI:http://dx.doi.org/10.7554/eLife.08378.015

Mentions: Methanocaldococcus jannaschii TFEα binds the RNAP in a bidentate fashion, the WH domain interacts with the tip of the RNAP clamp coiled coil while the ZR domain locates to the base of the clamp and the stalk module (Grohmann et al., 2011). In order to characterise the binding characteristics of TFEα/β to Sso RNAP we produced a recombinant RNAP clamp analogously to (Martinez-Rucobo et al., 2011). Gel filtration elution profiles show that TFEα forms a stable complex with the recombinant RNAP clamp since both proteins eluted in earlier fractions corresponding to a larger size (Figure 5). The elution of both proteins was slightly asymmetrical, possibly due to partial dissociation during chromatography. Similarly dimeric TFEα/β forms a stable complex with the clamp since all three proteins co-eluted in a symmetrical fashion. Deletion of the TFEα ZR domain (TFEα ΔZR, residues 114–147) leads to loss of complex formation. In contrast, deleting the TFEβ WH domain (TFEβ ΔWH, residues 1–73) does not impair binding (Figure 5) which suggests that the TFEβ WH domain does not contribute to RNAP binding.10.7554/eLife.08378.015Figure 5.TFEα/β forms a stable interaction with the RNAP clamp module.


Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39.

Blombach F, Salvadori E, Fouqueau T, Yan J, Reimann J, Sheppard C, Smollett KL, Albers SV, Kay CW, Thalassinos K, Werner F - Elife (2015)

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 SDS-PAGE and silver staining. The position of peaks for gel filtration marker proteins is indicated on top. For experiments with TFEα/β ΔWH the contrast was enhanced for the lower part of the gel in order to visualize TFEβ ΔWH.DOI:http://dx.doi.org/10.7554/eLife.08378.015
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495717&req=5

fig5: 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 SDS-PAGE and silver staining. The position of peaks for gel filtration marker proteins is indicated on top. For experiments with TFEα/β ΔWH the contrast was enhanced for the lower part of the gel in order to visualize TFEβ ΔWH.DOI:http://dx.doi.org/10.7554/eLife.08378.015
Mentions: Methanocaldococcus jannaschii TFEα binds the RNAP in a bidentate fashion, the WH domain interacts with the tip of the RNAP clamp coiled coil while the ZR domain locates to the base of the clamp and the stalk module (Grohmann et al., 2011). In order to characterise the binding characteristics of TFEα/β to Sso RNAP we produced a recombinant RNAP clamp analogously to (Martinez-Rucobo et al., 2011). Gel filtration elution profiles show that TFEα forms a stable complex with the recombinant RNAP clamp since both proteins eluted in earlier fractions corresponding to a larger size (Figure 5). The elution of both proteins was slightly asymmetrical, possibly due to partial dissociation during chromatography. Similarly dimeric TFEα/β forms a stable complex with the clamp since all three proteins co-eluted in a symmetrical fashion. Deletion of the TFEα ZR domain (TFEα ΔZR, residues 114–147) leads to loss of complex formation. In contrast, deleting the TFEβ WH domain (TFEβ ΔWH, residues 1–73) does not impair binding (Figure 5) which suggests that the TFEβ WH domain does not contribute to RNAP binding.10.7554/eLife.08378.015Figure 5.TFEα/β forms a stable interaction with the RNAP clamp module.

Bottom Line: The eukaryotic transcription factor TFIIE consists of α and β subunits.Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39.These activities are strictly dependent on the β subunit and the promoter sequence.

View Article: PubMed Central - PubMed

Affiliation: Institute for Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.

ABSTRACT
Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.

No MeSH data available.


Related in: MedlinePlus