News and Publications

Protein-Nucleic Acid Interactions in Transcriptional Regulation

J.M. Gottesfeld, L.A. Dickinson, J. Chacon, J.J. Long, S.J. McBryant, L. Neely, R. Winston, J. Xu, P.E. Wright, R.J. Gulizia,* D.E. Mosier,* P. Ghazal,* E.E. Baird,** J.W. Trauger,** P.B. Dervan**

* Department of Immunology, TSRI
** California Institute of Technology, Pasadena, CA

SEQUENCE-SPECIFIC INHIBITION OF GENE TRANSCRIPTION WITH DESIGNED LIGANDS

Our collaborators, P.B. Dervan and colleagues at the California Institute of Technology, have established rules for sequence-specific recognition of DNA in the minor groove with pyrrole-imidazole polyamides. These molecules have affinities and specificities for their target DNA sequences that are comparable to the affinities and specificities of natural DNA-binding transcriptional regulatory proteins. Moreover, these compounds are the only available class of small molecules that can bind predetermined DNA sequences. We have shown that these molecules are effective inhibitors of protein-DNA interactions and transcription both in vitro and in cells in culture. For genes encoding the small RNAs transcribed by RNA polymerase III, we found that polyamides can interfere with the DNA-binding activity of both general (TFIIIB) and gene-specific (TFIIIA) transcription factors.

We have investigated useful gene targets for polyamides with the aim of developing therapeutic agents for human diseases. Protein-coding genes use both gene- and tissue-specific transcription factors as well as general transcription factors for transcription by RNA polymerase II. Because the binding sites for these protein factors are found in numerous genes, polyamides have been designed to bind sequences adjacent to these binding sites, which tend to be unique for each gene.

We have developed a general method for gene-specific inhibition of basal transcription by RNA polymerase II. Many protein-coding genes contain TATA elements upstream from the transcription start site. This sequence is bound by the TATA-box binding protein subunit of the general transcription factor TFIID. A polyamide designed to recognize and bind the sequences adjacent to the HIV type 1 (HIV-1) TATA element can inhibit binding of the TATA-box binding protein and HIV-1 promoter-specific transcription. The generality of this approach has been confirmed by the successful inhibition of (1) interactions between several different TATA elements and the TATA-box binding protein and (2) transcription from other viral genes (human cytomegalovirus major immediate-early and polymerase genes) and a human cellular gene (the Her-2/neu oncogene involved in human breast cancer).

We have also used polyamides to inhibit the DNA-binding activity of transcriptional activator proteins. Polyamides were designed and synthesized to bind the sequences immediately adjacent to and overlapping the binding sites for the lymphoid enhancer factors LEF-1 and Ets-1 in the HIV-1 enhancer. These polyamides inhibited binding of LEF-1 and Ets-1 to HIV-1 DNA and inhibited LEF-1--dependent transcription in an in vitro transcription system. Ets-1 and another lymphoid factor, NF-B, bind HIV-1 proviral DNA in a cooperative manner. We found that a polyamide that directly inhibits Ets-1 binding also abolishes the cooperative association of NF-B with the HIV-1 enhancer.

The polyamides described have been used as inhibitors of HIV-1 replication in a cell culture assay system. D. Mosier and colleagues, Department of Immunology, found that individual polyamides cause limited (70--90%) inhibition of viral replication when used separately; however, a combination of 2 polyamides acts synergistically and causes highly effective (>99.9%) inhibition of replication. This inhibition is DNA sequence specific, because mismatch polyamides do not affect viral replication, and other cellular genes, which either do not contain polyamide-binding sites in their promoter elements or contain single nucleotide mismatches from the polyamide-binding sites in the HIV-1 promoter, are not inhibited by the polyamides. Of importance, no cytotoxic effects are associated with polyamide treatment. Taken together, these results indicate that polyamides can be designed to target sequences adjacent to the binding sites for general, gene-, and tissue-specific transcription factors and that these polyamides will be useful for the inhibition of gene-specific transcription in vivo, because polyamides appear to be cell-permeable agents. Our results suggest that these compounds will be useful as therapeutic agents in the treatment of human diseases, such as viral and other pathogenic diseases and cancer.

PROTEIN--NUCLEIC ACID INTERACTIONS

We have continued our studies on the DNA- and RNA-binding activity of the 5S RNA gene-specific transcriptional regulatory protein TFIIIA. Previous studies indicated that zinc fingers 1--3 of this 9-finger protein are largely responsible for DNA binding and that the central zinc fingers 4--7 are similarly responsible for binding 5S ribosomal RNA. We have determined the key region within the 5S RNA molecule critical for binding an RNA-specific zinc-finger peptide. Various structure- and sequence-specific nucleases are being used to examine the solution conformation of this selected RNA molecule, and nuclear magnetic resonance will be used for structural studies of both this molecule and the zinc-finger peptide. Additional protein-DNA binding studies concern determination of the amino acid residues of the Drosophila basic helix-loop-helix regulatory protein Deadpan that mediate protein dimerization.

TRANSCRIPTIONAL REGULATION OF CELL GROWTH AND DEVELOPMENT

Many aspects of transcriptional regulation in eukaryotic cells involve reversible protein phosphorylation events, such as the global repression of nuclear transcription that occurs when cells enter mitosis. The master mitotic protein kinase, cdc2/cyclin B, is sufficient to inhibit transcription by RNA polymerases II and III in both cellular extracts and reconstituted transcription systems. For class III transcription, the target of this protein kinase is the general transcription factor TFIIIB. For class II transcription, the general transcription factors TFIID and TFIIH are inactivated by the cdc2/cyclin B kinase. We are determining the polypeptide subunits of these factors that are phosphorylated and thereby inactivated by the mitotic kinase.

PUBLICATIONS

Long, J.J., Leresche, A., Kriwacki, R.W., Gottesfeld, J.M. Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis. Mol. Cell. Biol. 18:1467, 1998.

McBryant, S.J., Gottesfeld, J.M. Differential kinetics of transcription complex assembly distinguish oocyte and somatic 5S RNA genes of Xenopus. Gene Expr. 6:387, 1997.

Neely, L., Trauger, J.W., Baird, E.E., Dervan, P.B., Gottesfeld, J.M. Importance of minor groove binding zinc fingers within the transcription factor IIIA-DNA complex. J. Mol. Biol. 274:439, 1997.

Wuttke, D.S., Foster, M.P., Case, D.A., Gottesfeld, J.M., Wright, P.E. Solution structure of the first three zinc fingers of TFIIIA complexed with its cognate DNA sequence: Determinants of affinity and sequence specificity. J. Mol. Biol. 273:183, 1997.