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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

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 purification was raised to 500 mM KCl and a 20 CV wash with 50 mM imidazole was introduced prior to elution. The upper panel shows an SDS-gel with the Ni-affinity elution fractions with total protein stained with SYPRO Ruby. The lower panel shows the immunodetection with Sso0944 and TFEα antisera of the Ni-affinity elution fractions.DOI:http://dx.doi.org/10.7554/eLife.08378.004
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fig1s1: 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 purification was raised to 500 mM KCl and a 20 CV wash with 50 mM imidazole was introduced prior to elution. The upper panel shows an SDS-gel with the Ni-affinity elution fractions with total protein stained with SYPRO Ruby. The lower panel shows the immunodetection with Sso0944 and TFEα antisera of the Ni-affinity elution fractions.DOI:http://dx.doi.org/10.7554/eLife.08378.004

Mentions: We chose the hRPC39 homologue Sso0944 from the archaeon S. solfataricus (Sso) as model protein because the gene is a good representative of its kind (Blombach et al., 2009). To identify interaction partners of Sso0944 we expressed His-tagged Sso0944 in S. solfataricus M16 and probed the presence of co-purifying components of the basal transcription apparatus following metal-affinity chromatography by immunodetection. While we found no evidence that the RNAP, TBP or TFB1 co-purified with Sso0944, TFEα co-eluted with Sso0944, indicating that TFEα and Sso0944 are associated in vivo (Figure 1B). Sypro Ruby-stained SDS-PAGE of the affinity-purified material demonstrates that the polypeptides form a dimeric complex, that is, their association is not dependent on additional factors (Figure 1—figure supplement 1). To rule out the possibility that the affinity tag of Sso0944 prevented its stable association with the RNAP we fractionated a wild type S. solfataricus P2 cell lysate by size exclusion chromatography and analysed the fractions using immunodetection. The elution profile of endogenous Sso0944 overlapped with that of TFEα consistent with a heterodimeric TFEα/Sso0944 complex of 36.1 kDa. The elution profile of TFEα was somewhat broader indicating that part of TFEα might be present in the monomeric form. RNAP eluted in earlier fractions corresponding to its molecular weight of approximately 400 kDa (Figure 1C). As Sso0944 is not stably incorporated into RNAP in contrast to the related RNAPIII subunit hRPC39, but rather forms a complex with TFEα, we renamed the archaeal protein TFEβ.


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)

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 purification was raised to 500 mM KCl and a 20 CV wash with 50 mM imidazole was introduced prior to elution. The upper panel shows an SDS-gel with the Ni-affinity elution fractions with total protein stained with SYPRO Ruby. The lower panel shows the immunodetection with Sso0944 and TFEα antisera of the Ni-affinity elution fractions.DOI:http://dx.doi.org/10.7554/eLife.08378.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: 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 purification was raised to 500 mM KCl and a 20 CV wash with 50 mM imidazole was introduced prior to elution. The upper panel shows an SDS-gel with the Ni-affinity elution fractions with total protein stained with SYPRO Ruby. The lower panel shows the immunodetection with Sso0944 and TFEα antisera of the Ni-affinity elution fractions.DOI:http://dx.doi.org/10.7554/eLife.08378.004
Mentions: We chose the hRPC39 homologue Sso0944 from the archaeon S. solfataricus (Sso) as model protein because the gene is a good representative of its kind (Blombach et al., 2009). To identify interaction partners of Sso0944 we expressed His-tagged Sso0944 in S. solfataricus M16 and probed the presence of co-purifying components of the basal transcription apparatus following metal-affinity chromatography by immunodetection. While we found no evidence that the RNAP, TBP or TFB1 co-purified with Sso0944, TFEα co-eluted with Sso0944, indicating that TFEα and Sso0944 are associated in vivo (Figure 1B). Sypro Ruby-stained SDS-PAGE of the affinity-purified material demonstrates that the polypeptides form a dimeric complex, that is, their association is not dependent on additional factors (Figure 1—figure supplement 1). To rule out the possibility that the affinity tag of Sso0944 prevented its stable association with the RNAP we fractionated a wild type S. solfataricus P2 cell lysate by size exclusion chromatography and analysed the fractions using immunodetection. The elution profile of endogenous Sso0944 overlapped with that of TFEα consistent with a heterodimeric TFEα/Sso0944 complex of 36.1 kDa. The elution profile of TFEα was somewhat broader indicating that part of TFEα might be present in the monomeric form. RNAP eluted in earlier fractions corresponding to its molecular weight of approximately 400 kDa (Figure 1C). As Sso0944 is not stably incorporated into RNAP in contrast to the related RNAPIII subunit hRPC39, but rather forms a complex with TFEα, we renamed the archaeal protein TFEβ.

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