Protein-nucleic acid interactions are the fundamental basis for the control of genetic expression. Xenopus laevis contains two 5S rRNA multigene families that provide a simple, tractable model for the de- velopmental regulation of transcription by trans-acting factors. Our long term goal is to understand the temporal expression of these two gene families as a model for more complex developmental systems. The syn- thesis of 5S rRNA is primarily mediated by the zinc finger transcription factor TFIIIA which has the distinctive ability to bind to the 5S rRNA gene as a positive regulator of transcription and to the gene's transcript, 5S rRNA, to form a storage particle for the latter nucleic acid. We have concentrated on the ability of TFIIIA to bind to both nucleic acids since this is fundamental to the regulatory activity of the protein and because it provides a unique problem with regard to protein-nucleic acid recognition. The two TFIIIA-nucleic acid complexes have been crosslinked by UV irradiation. The sites of adduct formation on the protein will be determined in order to assess the relative importance of the nine fingers for binding to the different nucleic acids and whether the linker sequences that connect the fingers form important contacts to either nucleic acid. A combination of molecular biology and organic chemistry will be used to introduce a metal-binding amino acid analogue at specific sites within TFIIIA. The precise alignment of the factor along both the gene and 5S RNA will be mapped by exploiting the nucleic acid cleavage activity of the appended metal. These modified forms of the factor also will be used to study its interaction with the gene in the dynamic conditions of transcription when RNA polymerase moves through, but does not disrupt the transcription complex. Transition metal complexes that bind to RNA on the basis of shape complementarity will be used to delineate the tertiary organization of the molecule. Using specific mutants of the RNA, we will determine whether there is a correlation between the identified structures and recognition by TFIIIA. We have detected an interaction between ribosomes and the oocyte 5S rRNA genes. We will characterize this interaction to ascertain whether it is specific. We will determine whether it is related to the specific destabilization of transcription complexes on oocyte genes that occurs at meiosis. Chemical nucleases were employed to analyze the TFIIIA.5S rRNA particle. A similar analysis of the complex of ribosomal protein L5 with 5S rRNA will be undertaken. This affords the opportunity to examine the binding of two proteins, that exhibit no sequence identity, to congruent sites on an RNA molecule.
|Lambert, Lester J; Miller, Marvin J; Huber, Paul W (2015) Tetrahydrofuranyl and tetrahydropyranyl protection of amino acid side-chains enables synthesis of a hydroxamate-containing aminoacylated tRNA. Org Biomol Chem 13:2341-9|
|Malik, Mariam Q; Bertke, Michelle M; Huber, Paul W (2014) Small ubiquitin-like modifier (SUMO)-mediated repression of the Xenopus Oocyte 5 S rRNA genes. J Biol Chem 289:35468-81|
|Scripture, J Benjamin; Huber, Paul W (2011) Binding site for Xenopus ribosomal protein L5 and accompanying structural changes in 5S rRNA. Biochemistry 50:3827-39|
|Gellett, Amanda M; Huber, Paul W; Higgins, Pamela J (2008) Synthesis of the unnatural amino acid N-N-(ferrocene-1-acetyl)-l-lysine: a novel organometallic nuclease. J Organomet Chem 693:2959-2962|