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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

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. In parallel increasing amounts of recombinant dimeric TFEα/β (non-His-tagged) or TBP were loaded in order to generate a calibration curve (black crosses) that was used to determine the expression levels of TFEα, TFEβ and TBP (red diamond, average value, error bars show 1× standard deviation).DOI:http://dx.doi.org/10.7554/eLife.08378.005
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fig1s2: 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. In parallel increasing amounts of recombinant dimeric TFEα/β (non-His-tagged) or TBP were loaded in order to generate a calibration curve (black crosses) that was used to determine the expression levels of TFEα, TFEβ and TBP (red diamond, average value, error bars show 1× standard deviation).DOI:http://dx.doi.org/10.7554/eLife.08378.005

Mentions: In order to compare and characterise the steady-state levels of TFEα and TFEβ during exponential and stationary growth of S. solfataricus we carried out quantitative Western blotting. TFEα and TFEβ levels are near stoichiometric during exponential growth (24 ± 3 pmol/mg soluble protein and 27 ± 3 pmol/mg soluble protein, respectively) and about sevenfold lower than TBP levels (184 ± 17 pmol/mg) (Figure 1—figure supplement 2). TFEβ levels are decreased when cells enter stationary phase while RNAP, TBP, TFB and TFEα remain largely unaffected (Figure 1D). Our results demonstrate that dimeric TFEα/β is the predominant form of the factor in exponentially growing cells, and that the steady-state levels of the complex vary as a function of the growth cycle.


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)

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. In parallel increasing amounts of recombinant dimeric TFEα/β (non-His-tagged) or TBP were loaded in order to generate a calibration curve (black crosses) that was used to determine the expression levels of TFEα, TFEβ and TBP (red diamond, average value, error bars show 1× standard deviation).DOI:http://dx.doi.org/10.7554/eLife.08378.005
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495717&req=5

fig1s2: 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. In parallel increasing amounts of recombinant dimeric TFEα/β (non-His-tagged) or TBP were loaded in order to generate a calibration curve (black crosses) that was used to determine the expression levels of TFEα, TFEβ and TBP (red diamond, average value, error bars show 1× standard deviation).DOI:http://dx.doi.org/10.7554/eLife.08378.005
Mentions: In order to compare and characterise the steady-state levels of TFEα and TFEβ during exponential and stationary growth of S. solfataricus we carried out quantitative Western blotting. TFEα and TFEβ levels are near stoichiometric during exponential growth (24 ± 3 pmol/mg soluble protein and 27 ± 3 pmol/mg soluble protein, respectively) and about sevenfold lower than TBP levels (184 ± 17 pmol/mg) (Figure 1—figure supplement 2). TFEβ levels are decreased when cells enter stationary phase while RNAP, TBP, TFB and TFEα remain largely unaffected (Figure 1D). Our results demonstrate that dimeric TFEα/β is the predominant form of the factor in exponentially growing cells, and that the steady-state levels of the complex vary as a function of the growth cycle.

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