The TATA box-binding protein (TBP) is required by all three eukaryotic RNA polymerases for correct initiation of transcription of ribosomal, messenger, small nuclear, and transfer RNAs. The cocrystal structure of the C-terminal/core region of human TBP complexed with the TATA element of the adenovirus major late promoter has been determined at 1.9 A resolution. Structural and functional analyses of the protein-DNA complex are presented, with a detailed comparison to our 1.9-A-resolution structure ofArabidopsis thaliana TBP2 bound to the same TATA box.Eukaryotes have three distinct RNA polymerases (forms I, II, and III) that catalyze transcription of nuclear genes (1). Despite their structural complexity, these multisubunit enzymes require sets of auxiliary proteins known as general transcription initiation factors to initiate transcription from corresponding class I, II, and III nuclear gene promoters (2)(3)(4)(5) We now report the crystal structure of human C-terminal/ core TBP (hTBPc) recognizing the AdMLP TATA element. The 1.9-A-resolution structure permits detailed structural and functional analyses of the protein-DNA interactions and provides further insights into how TBPs from different organisms recognize and direct transcription initiation from TATAcontaining promoters. MATERIALS AND METHODSCrystallization. hTBPc residues 155-335 (15) fused with an additional 20 N-terminal amino acids (MGSSHHHHHHSS-GLVPRGSH) was overexpressed in Escherichia coli [BL21-(DE3)pLysS] using the T7 RNA polymerase system (16). Cells grown at 30°C to an absorbance of 0.7 at 595 nm and induced with 0.5 mM isopropyl P3-D-thiogalactopyranoside for 3 h were harvested by low-speed centrifugation. Lysis was performed by three cycles of freeze-thaw and DNA digestion was performed by DNase I. The soluble fraction was applied directly to a Ni2+ ion affinity column and washed with a buffer containing increasing amounts of imidazole. hTBPc with an estimated homogeneity of 95% was then eluted from the resin with a buffer containing 100 mM EDTA. After overnight dialysis, removal of the histidine-containing N-terminal sequence was Abbreviations: AdMLP, adenovirus major late promoter; PIC, preinitiation complex; TBP, TATA box-binding protein; hTBPc, human core TATA box-binding protein; TBP2, TATA box-binding protein isoform 2; pol II, RNA polymerase II; rmsd, rms deviation.Data deposition: The atomic coordinates and structure factor amplitudes have been deposited in the Protein
The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites. To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension. Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site. Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15. By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription. The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site. Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites. The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand. Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment. Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts. Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.
Chromosomes in the eukaryotic nucleus are highly compacted. However, for many functional processes, including transcription initiation, the pairwise motion of distal chromosomal elements such as enhancers and promoters is essential and necessitates dynamic fluidity. Here, we used a live-imaging assay to simultaneously measure the positions of pairs of enhancers and promoters and their transcriptional output while systematically varying the genomic separation between these two DNA loci. Our analysis reveals the coexistence of a compact globular organization and fast subdiffusive dynamics. These combined features cause an anomalous scaling of polymer relaxation times with genomic separation leading to long-ranged correlations. Thus, encounter times of DNA loci are much less dependent on genomic distance than predicted by existing polymer models, with potential consequences for eukaryotic gene expression.
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