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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

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 corresponding to hRPC39 (yellow) and hRPC39 + [4Fe-4S] (red) are observed (bottom and Figure 2—source data 1).DOI:http://dx.doi.org/10.7554/eLife.08378.011
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fig2s2: 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 corresponding to hRPC39 (yellow) and hRPC39 + [4Fe-4S] (red) are observed (bottom and Figure 2—source data 1).DOI:http://dx.doi.org/10.7554/eLife.08378.011

Mentions: In order to carry out a structure-function analysis of TFEα/β we expressed and purified a recombinant form of the TFEα/β complex in in Escherichia coli. Concentrated recombinant TFEα/β has a dark brown colour and its absorption spectrum displays a shoulder at 410 nm that is characteristic for iron-sulphur cluster harbouring proteins (Figure 2A). In order to define the origin of this absorption more precisely we recorded continuous-wave electron paramagnetic resonance (cw-EPR) spectra. The profile of the cw-EPR spectrum with a g-value of 2.01, its sensitivity to the reducing agent sodium dithionite and the sharp decrease in signal at temperatures above 20 K are consistent with a signal corresponding to a cubane [3Fe-4S]+ cluster (Figure 2B and Figure 2—figure supplement 1). Double integration and comparison with a spin standard indicate a low cluster-occupancy of approximately 4%, which is in contrast to the strong 410 nm signal in the absorption spectrum. It is common that before reduction aerobically prepared proteins containing [4Fe-4S] clusters display a [3Fe-4S]+ signal due to oxidative impairment (Beinert et al., 1996). Addition of a reducing agent often results in the disappearance of the signal of the [3Fe-4S]+ cluster concomitant with the appearance of the signal from the intact [4Fe-4S]+ cluster. Here, however, only the former effect is observed: [4Fe-4S]2+ clusters may not be reduced—and remain EPR-silent—if the redox potential is lower than that of the reducing agent (dithionite), or if the rate of reduction is too slow. Alternatively the reduced [4Fe-4S]+ cluster could be present in a high spin state, which could give rise to a signal too broad to be detected. To investigate the presence of an EPR-silent [4Fe-4S]+ cluster, we recorded native mass spectra (MS) of TFEα/β (Figure 2C and Figure 2—source data 1). The spectrum contained two major species one with a mass of 21,236.6 Da and the other with a mass of 36,530.8 Da. The former corresponds to the expected mass for TFEα with a Zn2+ ion bound by its zinc ribbon (ZR) domain while the later corresponds to the mass of the TFEα/β harbouring a Zn2+ ion and a [4Fe-4S]2+ cluster. Given that the TFEα ZR harbours a Zn2+ ion, the [4Fe-4S]2+ cluster must be coordinated by the four cysteines in the C-terminal domain of TFEβ. As this domain is highly conserved in human hRPC39 (Figure 2G), we examined the human protein for the presence of an equivalent FeS cluster. Similar to archaeal TFEβ, the human hRPC62/C39 complex shows an absorption spectrum with a shoulder at 410 nm (Figure 2D); its cw-EPR signature is characteristic for a [3Fe-4S]+ cluster with low occupancy (<3%) (Figure 2E and Figure 2—figure supplement 1). The native mass spectrum this time revealed three major species with masses of 35,555.7, 62,516.3 and 98,455.5 Da corresponding to hRPC39, hRPC62 and hRPC62/C39 complex bound to a [4Fe-4S]2+ respectively (Figure 2F). Tandem MS experiments revealed that the cluster was bound to hRPC39 (Figure 2—figure supplement 2 and Figure 2—source data 1).10.7554/eLife.08378.008Figure 2.The C-terminal domain of Sso TFEβ harbours a 4Fe-4S cluster that is conserved in human RNAPIII subcomplex hRPC62/39.


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)

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 corresponding to hRPC39 (yellow) and hRPC39 + [4Fe-4S] (red) are observed (bottom and Figure 2—source data 1).DOI:http://dx.doi.org/10.7554/eLife.08378.011
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Related In: Results  -  Collection

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fig2s2: 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 corresponding to hRPC39 (yellow) and hRPC39 + [4Fe-4S] (red) are observed (bottom and Figure 2—source data 1).DOI:http://dx.doi.org/10.7554/eLife.08378.011
Mentions: In order to carry out a structure-function analysis of TFEα/β we expressed and purified a recombinant form of the TFEα/β complex in in Escherichia coli. Concentrated recombinant TFEα/β has a dark brown colour and its absorption spectrum displays a shoulder at 410 nm that is characteristic for iron-sulphur cluster harbouring proteins (Figure 2A). In order to define the origin of this absorption more precisely we recorded continuous-wave electron paramagnetic resonance (cw-EPR) spectra. The profile of the cw-EPR spectrum with a g-value of 2.01, its sensitivity to the reducing agent sodium dithionite and the sharp decrease in signal at temperatures above 20 K are consistent with a signal corresponding to a cubane [3Fe-4S]+ cluster (Figure 2B and Figure 2—figure supplement 1). Double integration and comparison with a spin standard indicate a low cluster-occupancy of approximately 4%, which is in contrast to the strong 410 nm signal in the absorption spectrum. It is common that before reduction aerobically prepared proteins containing [4Fe-4S] clusters display a [3Fe-4S]+ signal due to oxidative impairment (Beinert et al., 1996). Addition of a reducing agent often results in the disappearance of the signal of the [3Fe-4S]+ cluster concomitant with the appearance of the signal from the intact [4Fe-4S]+ cluster. Here, however, only the former effect is observed: [4Fe-4S]2+ clusters may not be reduced—and remain EPR-silent—if the redox potential is lower than that of the reducing agent (dithionite), or if the rate of reduction is too slow. Alternatively the reduced [4Fe-4S]+ cluster could be present in a high spin state, which could give rise to a signal too broad to be detected. To investigate the presence of an EPR-silent [4Fe-4S]+ cluster, we recorded native mass spectra (MS) of TFEα/β (Figure 2C and Figure 2—source data 1). The spectrum contained two major species one with a mass of 21,236.6 Da and the other with a mass of 36,530.8 Da. The former corresponds to the expected mass for TFEα with a Zn2+ ion bound by its zinc ribbon (ZR) domain while the later corresponds to the mass of the TFEα/β harbouring a Zn2+ ion and a [4Fe-4S]2+ cluster. Given that the TFEα ZR harbours a Zn2+ ion, the [4Fe-4S]2+ cluster must be coordinated by the four cysteines in the C-terminal domain of TFEβ. As this domain is highly conserved in human hRPC39 (Figure 2G), we examined the human protein for the presence of an equivalent FeS cluster. Similar to archaeal TFEβ, the human hRPC62/C39 complex shows an absorption spectrum with a shoulder at 410 nm (Figure 2D); its cw-EPR signature is characteristic for a [3Fe-4S]+ cluster with low occupancy (<3%) (Figure 2E and Figure 2—figure supplement 1). The native mass spectrum this time revealed three major species with masses of 35,555.7, 62,516.3 and 98,455.5 Da corresponding to hRPC39, hRPC62 and hRPC62/C39 complex bound to a [4Fe-4S]2+ respectively (Figure 2F). Tandem MS experiments revealed that the cluster was bound to hRPC39 (Figure 2—figure supplement 2 and Figure 2—source data 1).10.7554/eLife.08378.008Figure 2.The C-terminal domain of Sso TFEβ harbours a 4Fe-4S cluster that is conserved in human RNAPIII subcomplex hRPC62/39.

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