Abstract
Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk.
Highlights
Functional cross-talk between the promoter and terminator of a gene has long been noted
transcription factor IIB (TFIIB) is a multidomain protein that is composed of an N-terminal zinc ribbon domain for polymerase II (pol II) binding [1,2,3,4], a finger/reader domain for transcription start site selection and de novo transcription initiation (1, 4 – 6), and two C-terminal cyclin-like domains that form a ternary complex with the TATA box DNA and TATA box– binding protein (TBP) [7, 8] (Fig. 1A)
Our structure reveals an unprecedented degree of plasticity of TFIIBc, we note that the conformations of TFIIBc from our structure, like all crystal structures, are partially influenced by crystal packing
Summary
Functional cross-talk between the promoter and terminator of a gene has long been noted. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We propose a hand-off model in which Ssu mediates a conformational transition in TFIIB, accounting for the role of Ssu in transcription reinitiation, gene looping, and promoter-terminator cross-talk. One mechanism of transcriptional activation suggests that VP16AD displaces the intramolecular interaction between the N-terminal region of TFIIB (TFIIBn) and the cyclin-like domain-containing core region (TFIIBc), converting TFIIB to an active conformation for PIC assembly (16 –21). Ssu binds to the N-terminal region of an RNA-processing
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