DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules

Abstract

The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.

Figure 1

(A) Probable DNA contacts made by the first three HMG boxes of Xenopus UBF. A dimer of Xenopus UBF is shown with each HMG box interacting with adjacent 20 bp segments of DNA. (B) A model explaining the role of in-phase DNA bending by the HMG boxes of UBF in formation of the enhancesome. A view face on to the DNA loop and another edge on to the DNA loop are shown. In both (A) and (B) the DNA is indicated in red as a (bent) rod and the ‘core’ region of Xenopus UBF is shown in blue.

Figure 2

(A) The domain structure of Xenopus UBF and the C-terminally truncated ‘core’ UBF, Nbox13. The sequence of the peptide linking the N-terminal dimerisation domain (Dimer.) and HMG box 1 is shown aligned with the N-terminal sequence of NHP6a, known to bind the narrowed major groove on the inside of the HMG box-induced DNA bend. Basic amino acids are shown in black and similarities between the sequences are boxed. (B) The wild-type (WT) and mutant (‘Minus’ or ‘Plus,) linkers used in the present study. (C) Analysis of the protein masses of complexes of Nbox13 (Minus and Plus mutants) bound to the 1.1 kb ribosomal enhancer DNA repeat from X.laevis (19) as determined by ESI. (D) Dimerisation of the free Core UBF mutants. [³⁵S]methionine-labelled FLAG-tagged forms of wild-type and each core UBF mutant (Applied) were allowed to interact with the immobilised equivalent UBF form (Matrix). The protein retained by homodimerisation was analysed by SDS–PAGE followed by phosphorimaging (see Materials and Methods). Relative homodimerisation (Rel. Dimer) is given as recovery of bound ³⁵S-labelled mutant protein relative to wild-type.

Figure 3

Minus and Plus mutations of core UBF prevent the formation of the 360° DNA loop characteristic of the wild-type enhancesome. (A) Dimers of Minus and Plus core UBF mutants complexed with DNA. (B) Typical enhancesome complexes containing dimers of wild-type core UBF complexed to DNA. (A) and (B) show superimposed phosphorus (DNA in red) and total mass (grey) ESI images of wild-type and core UBF mutants bound to the 1.1 kb Xenopus ribosomal enhancer DNA. The calculated protein mass for each complex is given.

Figure 4

Monomer complexes of Minus and Plus core UBF mutants bound to the 1.1 kb Xenopus ribosomal enhancer DNA. (A) Monomer complexes formed by the Minus core UBF mutant. (B) Monomer Plus core UBF mutant complexes. Phosphorus and total mass images are shown superimposed as in Figure 3. The calculated protein mass for each complex is given.

Figure 5

Monomer Minus and Plus core UBF mutant complexes were analysed for DNA bend angle associated with the complex. (A) A typical bend angle measurement on a monomer complex. (B) Histogram of bend angles observed for both Minus and Plus mutants. The angle expected for the wild-type enhancesome is given.

Figure 6

Rethinking the enhancesome model to account for the length of interdomain peptide linkers. (A) The structure of a typical sequence non-specific HMG box fold, that of HMG-D (32). The three helices and the N- and C-termini (NH and COOH) are indicated. Note that the polypeptide chain enters and exits the HMG box fold proximally. (B) A schematic of the core enhancesome indicating the probable positions and orientations of the HMG boxes. The length of peptide linkers is given in amino acids (aa), the linkers themselves are indicated by broad arrows N- to C-terminally. Previous data shows that boxes 1 and 2 bind adjacent DNA sequences, enforcing an inversion of box 1 relative to box 2 and, hence, a head-to-head topology for these two boxes. The DNA is shown as a bent red ribbon and the HMG boxes are shown in blue using the HMG-D structure.