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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.


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 indicated. A recovery marker (r.m.) was included. The lower panels show the quantifications of synthesized transcript.DOI:http://dx.doi.org/10.7554/eLife.08378.019
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fig7s1: 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 indicated. A recovery marker (r.m.) was included. The lower panels show the quantifications of synthesized transcript.DOI:http://dx.doi.org/10.7554/eLife.08378.019

Mentions: In order to ascertain the function of TFEα/β on productive transcription we fused different promoters to a C-less cassette and carried out transcription assays in the presence of GTP, ATP and UTP. We compared the viral T6 and five cellular promoters of protein encoding genes (Sso EF-1α, Sso SSB, and Sso Rpo5), as well as noncoding RNA genes (Sso tRNALeu and 16S/23S rRNA) (Figure 7B). On all tested promoters the addition of TFEα/β stimulated transcription by approximately twofold to fourfold, with the strong T6 and 16S/23S rRNA promoters showing a weaker response (Figure 7C and Figure 7—figure supplement 1). These results suggest that the stimulation of transcription by TFEα/β is dependent on the sequence of the promoter. Considering that TFEα/β stimulates DNA melting we hypothesised that the initially melted region of the promoter (−12 to +4) determines the amplitude of the stimulation. We generated hybrid promoters encompassing the TATA-box and surrounding region (position −46 to −13) of the weakly stimulated 16S/23S rRNA promoter with the initially melted and transcribed regions of the stronger stimulated Sso tRNALeu (−12 to +5) or EF-1α promoters (−12 to +7). In absence of TFEα/β the hybrid promoters show reduced activity compared to the wild-type rRNA promoter underlining that the initially melted region contributes to the strength of the ribosomal promoter (Figure 7D). In line with our hypothesis TFEα/β stimulated transcription on the two hybrid promoters to greater extent when compared to the wild-type rRNA promoter, confirming that the sequence of the initially melted region determines the extent of TFEα/β stimulation. Interestingly, both TFEβ WH deletion and charge reversal mutations, and the TFEα ZR deletion mutants were able to stimulate transcription from the T6 and Rpo5 promoters (Figure 7E and Figure 7—figure supplement 2).


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)

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 indicated. A recovery marker (r.m.) was included. The lower panels show the quantifications of synthesized transcript.DOI:http://dx.doi.org/10.7554/eLife.08378.019
© Copyright Policy
Related In: Results  -  Collection

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

fig7s1: 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 indicated. A recovery marker (r.m.) was included. The lower panels show the quantifications of synthesized transcript.DOI:http://dx.doi.org/10.7554/eLife.08378.019
Mentions: In order to ascertain the function of TFEα/β on productive transcription we fused different promoters to a C-less cassette and carried out transcription assays in the presence of GTP, ATP and UTP. We compared the viral T6 and five cellular promoters of protein encoding genes (Sso EF-1α, Sso SSB, and Sso Rpo5), as well as noncoding RNA genes (Sso tRNALeu and 16S/23S rRNA) (Figure 7B). On all tested promoters the addition of TFEα/β stimulated transcription by approximately twofold to fourfold, with the strong T6 and 16S/23S rRNA promoters showing a weaker response (Figure 7C and Figure 7—figure supplement 1). These results suggest that the stimulation of transcription by TFEα/β is dependent on the sequence of the promoter. Considering that TFEα/β stimulates DNA melting we hypothesised that the initially melted region of the promoter (−12 to +4) determines the amplitude of the stimulation. We generated hybrid promoters encompassing the TATA-box and surrounding region (position −46 to −13) of the weakly stimulated 16S/23S rRNA promoter with the initially melted and transcribed regions of the stronger stimulated Sso tRNALeu (−12 to +5) or EF-1α promoters (−12 to +7). In absence of TFEα/β the hybrid promoters show reduced activity compared to the wild-type rRNA promoter underlining that the initially melted region contributes to the strength of the ribosomal promoter (Figure 7D). In line with our hypothesis TFEα/β stimulated transcription on the two hybrid promoters to greater extent when compared to the wild-type rRNA promoter, confirming that the sequence of the initially melted region determines the extent of TFEα/β stimulation. Interestingly, both TFEβ WH deletion and charge reversal mutations, and the TFEα ZR deletion mutants were able to stimulate transcription from the T6 and Rpo5 promoters (Figure 7E and Figure 7—figure supplement 2).

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.