The Role of Transcription Factor B in Transcription Initiation and Promoter Clearance in the Archaeon Sulfolobus acidocaldarius

Stephen D Bell,Stephen P Jackson

doi:10.1074/jbc.275.17.12934

Abstract

Mechanisms of transcription initiation appear to be remarkably conserved between archaea and eucaryotes. For instance, there is homology between archaeal and eucaryotic basal transcription factors. Also, the archaeal RNA polymerase (RNAP) resembles eucaryotic nuclear RNAPs in subunit composition and at the amino acid sequence level. Here, we examine the role of transcription factor B, the archaeal homologue of eucaryotic transcription factor IIB, in transcription initiation. We show that the N-terminal region of transcription factor B is required for RNAP recruitment. Furthermore, we reveal that mutation of a conserved residue immediately C-terminal of the N-terminal zinc ribbon motif abrogates transcription on certain promoters. Finally, we identify the promoter sequences responsive to this mutation and demonstrate that the effect of the mutation is to block a late stage in transcription initiation, following formation of the promoter open complex.

Highlights

Transcription in archaea requires a complex multisubunit RNA polymerase (RNAP)1 and two transcription factors, the archaeal TATA box-binding protein and transcription factor B (TFB). aTBP is a homologue of the eucaryotic TATA box-binding protein (TBP), and TFB is homologous to eucaryotic TFIIB
While this work was in progress, the sequence of a S. acidocaldarius box Abinding protein was deposited in the GenBankTM/EBI Data Bank,2 and it is 100% identical to the saTBP we have cloned. saTBP is 92% identical to its S. shibatae homologue, whereas saTFB is 89% identical to S. shibatae TFB
Whereas the saTFB gene has a conventional ATG start codon, the presumptive translation product of the saTBP gene starts with an Ile that is encoded by an ATT codon

Summary

Introduction

Transcription in archaea requires a complex multisubunit RNA polymerase (RNAP)1 and two transcription factors, the archaeal TATA box-binding protein (aTBP) and transcription factor B (TFB) (for reviews, see Refs. 1 and 2). aTBP is a homologue of the eucaryotic TATA box-binding protein (TBP), and TFB is homologous to eucaryotic TFIIB. Addition of highly purified saRNAP to the ternary complex caused a considerable extension of the footprint on the downstream side of the TATA box, extending to ϩ8 with respect to the transcription start site (Fig. 2B). Ternary complexes containing TBP and either saTFB or saTFB-E46K were effective at recruiting RNAP to the promoter.

Results

Conclusion