Paul Huber - US grants

The developmental control of the expression of Xenopus 5S rRNA genes is mediated by the activitiy of an exceptional protein called transcription factor IIIA (TFIIIA). This protein has the distinctive ability to bind to both the 5S gene and its transcript, 5S rRNA. TFIIIA as well as the whole collection of proteins that control the activity of oocyte and somatic 5S genes are the prototype for the study of developmental regulation of gene expression and determination. The identification of the binding site for TFIIIA on 5S rRNA has led us to propose that the protein utilizes similar contacts in binding to either nucleic acid. We will test this proposal in two ways. Using circular dichroism spectroscopy we will determine the conformation of the DNA when TFIIIA binds to the 5S gene. If the interaction of the protein with the two nucleic acids is similar then the DNA will have to be in the A conformation in order to form a structure comparable to 5S rRna. We will carry out an extensive series of site-directed mutagenesis experiments to make changes in both the gene and the transcript. We will determine whether a particular change has a similar effect on the binding of TFIIIA to both nucleic acids. The binding site on 5S rRNA for TFIIIA closely approximates that for ribosomal protein L5. This suggests that the two proteins may be related; at least there may be homology between their nucleic acid binding domains. We will measure the binding of L5 and TFIIIA to the variant 5S rRNAs synthesized in the mutagenesis experiments to compare the interactions of the proteins with their cognate nucleic acid. We will test the homology of the proteins by immunochemical techniques. We will prepare polyclonal antibodies to a proteolytic fragment of TFIIIA that contains the nucleic acid binding domain of the protein. We will determine whether the antiserum crossreacts with L5. If the result is positive, we will use the antiserum to locate the nucleic acid binding domain within L5. The unique properties of TFIIIA allow us to make certain invaluable comparisons: between the structures of two nucleic acids that are recognized by this protein, and between two proteins that recognize the same binding site on 5S rRNA.

biomedical equipment purchase;

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.

What we will do: Five levels of hierarchical self-assembly will be used to control the placement of single nanoparticles and inorganic molecules over areas that are hundreds of microns in size. Self-assembly and enzymatic processing steps will be used to create
DNA tiles

1 and multi-tile "rafts" that have dimensions in the 4-60 nm size range. The tiles will contain derivatization sites at known spatial locations to permit attachment of non-DNA components. Up to six different molecules or nanoparticles could be attached to each DNA tile. Molecular liftoff

2 will be used to direct the binding of the DNA rafts to lithographic features, such as 30 nm lines. The dimensions of the DNA rafts are similar to the dimensions of the lithographic features, so individual molecules that are attached to the rafts will be placed on the surface with great control and could be located near other lithographic structures. These capabilities would be very useful for construction of molecular quantum-dot cellular automata circuits and other molecular electronic devices.

Intellectual Merit: This project will: -explore hierarchical design as a tool for creation of supramolecular complexity -extend molecular liftoff, which has previously been used only for small inorganic molecules, to biomolecules such as DNA. -integrate top-down and bottom-up approaches to the fabrication of structures on the nanometer to micron size scale

Broader impact: This proposal combines detailed control over local physical structure with ultra-high resolution nanolithography to create non-repetitive arrays of the types required for large-scale implementation of different architectures for molecular electronics.

3 DNA will be used as a self-assembling circuit board for active components, which could include nanoparticles, other biomolecules, and small organic or inorganic molecules. This method could be used to construct technologically useful devices, such as molecular electronic field-programmable gate arrays that are integrated with I/O structures on a silicon chip.