TAF1B is a TFIIB-like component of the basal transcription machinery for RNA polymerase I

Abstract

Transcription by eukaryotic RNA polymerases (Pols) II and III and archaeal Pol requires structurally related general transcription factors TFIIB, Brf1, and TFB, respectively, which are essential for polymerase recruitment and initiation events. A TFIIB-like protein was not evident in the Pol I basal transcription machinery. We report that TAF1B, a subunit of human Pol I basal transcription factor SL1, is structurally related to TFIIB/TFIIB-like proteins, through predicted amino-terminal zinc ribbon and cyclin-like fold domains. SL1, essential for Pol I recruitment to the ribosomal RNA gene promoter, also has an essential postpolymerase recruitment role, operating through TAF1B. Therefore, a TFIIB-related protein is implicated in preinitiation complex assembly and postpolymerase recruitment events in Pol I transcription, underscoring the parallels between eukaryotic Pol I, II, and III and archaeal transcription machineries.

Fig 1

HsTAF1B domain organization and predicted secondary structures, derived from HHpred alignment of HsTAF1B and HsTFIIB (Fig. S1). HHpred is a sequence alignment method based on the pairwise comparison of profile Hidden Markov Models (HMMs) which facilitates protein remote homologue detection and structure prediction. (h denotes an α-helix).

Fig 2

The N-terminal Zn-ribbon of TAF1B has a post-recruitment role in transcription. (A) SL1 activity of in vivo-assembled, tag-immuno-affinity purified complexes from cells exogenously-expressing tagged TAF1B WT (lane 1), Zn-ribbon mutants C13A (lane 2), C31A (lane 3) or C34A (lane 4); in vitro transcription reactions included an immobilised human rDNA promoter template (IT-rDNA), Pol I and UBF. (B) Composition of in vivo-assembled SL1 complexes (immunodetection). (C) Binding of the rDNA promoter template by SL1 complexes, isolated as in A, containing WT TAF1B (lane 3), C13A (lane 4), purified endogenous SL1 (lane 1) (immunodetection). Control, no TAF1B expressed (lane 2). (D) Binding of Pol I to SL1 complexes containing WT TAF1B (lane 2) or C13A (lane 3) (immunodetection of Pol I subunit A190). Control (lane 1) as in C. (E) Initiation-competency of Pol I recruited to the rDNA promoter by SL1 complexes containing WT TAF1B (lanes 1 and 3) or C13A (lanes 2 and 4) (transcription assay +/− SL1, no added Pol I). IT-rDNA is defined in A.