[show abstract][hide abstract] ABSTRACT: Xenopus UBF (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. These include a short basic region adjacent to a dimer motif plus five high-mobility-group (HMG) boxes. All of these DNA-binding motifs exhibit low sequence specificity, whether assayed singly or together. In contrast, the HMG boxes recognize DNA structure that is formed when two double helices are crossed over each other. HMG box 1, in particular, requires association of two double helices before it will bind and, either by itself or in the context of the intact protein, will loop DNA and organize it into higher-order structures. We discuss how this mode of binding affects the function of xUBF as a transcription factor.
Molecular and Cellular Biology 06/1994; 14(5):2871-82. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5' terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an Internal Ribosome Entry Site (IRES) into the 5' leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5' trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.
Nucleic Acids Research 08/1993; 21(15):3451-7. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Xenopus laevis upstream binding factor (xUBF) is an RNA polymerase I transcription factor that is required for formation of the stable initiation complex. The 701-amino-acid protein contains three regions of homology to the chromosomal protein HMG1 (the HMG boxes), which act in comparative independence to cause DNA binding. DNA binding is augmented by a 102-residue amino-terminal domain that causes xUBF to form dimers. The dimerization domain is bipartite in structure, consisting of two regions with the potential to form amphipathic helices, separated by a gap of at least 22 amino acids. The carboxyl half of xUBF is relatively dispensable for transcription (including an 87-residue acidic tail). However, either altering the number of HMG boxes or interfering with dimerization eliminates transcription. The gap region of the dimerization domain is dispensable for dimerization but is absolutely required for transcription. This suggests that the gap region has a critical function in transcription distinct from any effect on dimerization or DNA binding.
Genes & Development 12/1991; 5(11):1957-68. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: We show that three protein fractions are required for accurate transcription initiation at a Xenopus laevis ribosomal gene promoter in vitro: RNA polymerase I, Rib1 and xUBF. The Rib1 and xUBF fractions are both necessary and sufficient for formation of a stable initiation complex. The xUBF fraction can be completely replaced by recombinant xUBF. We also report the sequence of a cDNA clone for xUBF. xUBF is 701 amino acids in length, contains domain which are related to a domain found in chromosomal proteins HMG 1 and 2, and has an acidic carboxy terminus of 87 amino acids. xUBF is closely similar in amino acid sequence to its previously reported human homolog, hUBF, except that xUBF has only three of the HMG-related domains while hUBF has four and therefore is 63 amino acids longer than xUBF.
The EMBO Journal 09/1991; 10(8):2297-303. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: On the ribosomal genes of Xenopus laevis, the T3 terminator is located approximately 60 bp upstream of the 5' boundary of the gene promoter. We have shown previously that mutation of the terminator simultaneously abolishes termination and impairs initiation by RNA polymerase I. Here, we show that the terminator influences the promoter by two distinct mechanisms. In one mechanism the terminator protects the promoter by preventing polymerase from reading through the initiation complex. In a second mechanism, the terminator interacts directly with the promoter, whether or not termination occurs. This positive interaction requires precise positioning of the terminator relative to the promoter and is sensitive to movement of the terminator by as little as 1 or 2 bp. We conclude that the terminator and promoter interact as one interdependent complex.
Genes & Development 08/1990; 4(7):1240-51. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe a partially fractionated in vitro transcription system from Xenopus laevis for the assay of transcription termination by RNA polymerase I. Termination in vitro was found to require a specific terminator sequence in the DNA and a DNA-binding protein fraction that produces a footprint over the terminator sequence.
Molecular and Cellular Biology 07/1990; 10(6):2793-800. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: We purified xUBF on the basis of its ability to specifically bind the enhancer elements of the Xenopus laevis rRNA genes. xUBF also binds to both upstream and downstream regions of the X. laevis ribosomal gene promoter and is essential for polymerase I transcription. Unexpectedly, xUBF binds to both upstream and downstream regions of the human ribosomal gene promoter, producing footprints that are indistinguishable from the footprints produced by hUBF, a previously described polymerase I transcription factor isolated from human cells. Despite extensive sequence divergence of vertebrate polymerase I promoters, these data suggest an evolutionary conservation of the primary DNA-protein interaction.
Genes & Development 12/1989; 3(11):1779-88. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have assayed a series of linker scanner mutants which cover the Xenopus laevis ribosomal gene promoter at approximately ten base pair intervals. All of these mutations adversely affect promoter activity with the exception of one mutation which stimulates activity. Thus, none are neutral. We show that most of the mutations can be partially rescued by ligating a block of enhancer elements upstream of the promoter. In addition, we have made extracts from liver nuclei which produce DNaseI protection footprints over the promoter. Analysis of both strands reveals a prominent footprinting domain from about -5 to -30. However, lesser changes in the digestion pattern are detected over most of the promoter. Previously published analyses have suggested that this promoter might be composed of three functional domains. The experiments presented here suggest that either 1) the three putative domains are so closely arranged that the boundaries are difficult to discern, or 2) the situation is more complex.
Nucleic Acids Research 10/1987; 15(18):7429-41. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: On the Xenopus laevis ribosomal genes, RNA polymerase traverses the entire repeating unit of gene plus spacer and terminates just upstream of the next gene promoter. A conserved 7 bp element located at about -200 is an essential part of this terminator. In this paper we show that, in addition to its termination function, this same sequence motif acts as an upstream element of the adjacent promoter and appears to contribute to the long-term stability of the transcription complex.