The C31 subunit belongs to a complex of three subunits (C31, C34 and C82) specific to RNA polymerase (pol) III that have no counterparts in other RNA polymerases. This complex is thought to play a role in transcription initiation since it interacts with the general initiation factor TFIIIB via subunit C34. We have obtained a conditional mutation of pol III by partially deleting the acidic C‐terminus of the C31 subunit. A Saccharomyces cerevisiae strain carrying this truncated C31 subunit is impaired in in vivo transcription of tRNAs and failed to grow at 37 degrees C. This conditional growth phenotype was suppressed by overexpression of the gene coding for the largest subunit of pol III (C160), suggesting an interaction between C160 and C31. The mutant pol III enzyme transcribed non‐specific templates at wild‐type rates in vitro, but was impaired in its capacity to transcribe tRNA genes in the presence of general initiation factors. Transcription initiation, but not termination or recycling of the enzyme, was affected in the mutant, suggesting that it could be altered on interaction with initiation factors or on the formation of the open complex. Interestingly, the C‐terminal deletion was also suppressed by a high gene dosage of the DED1 gene encoding a putative helicase.

The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear targeting of proteins. We have developed a chemical strategy for covalent coupling of NLS peptides to plasmid DNA. A p-azido-tetrafluoro-benzyl-NLS peptide conjugate was synthesized. This conjugate was used to covalently associate NLS peptides to plasmid DNA by photoactivation. Reporter gene was expressed after transfection of the plasmid-NLS conjugates in NIH 3T3 cells. The conjugates interacted specifically with the NLS-receptor importin alpha, but plasmid-NLS conjugates were not detected in the nucleus, by fluorescence microscopy, after cytoplasmic microinjection.

Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.

The alpha‐amanitin domain or domain f of the largest subunit of RNA polymerases is one of the most conserved of these enzymes. We have found that the C‐terminal part of domain f can be swapped between yeast RNA polymerase II and III. An extensive mutagenesis of domain f of C160, the largest subunit of RNA polymerase III, was carried out to better define its role and understand the mechanism through which C160 participates in transcription. One mutant enzyme, C160–270, showed much reduced transcription of a non‐specific template at low DNA concentrations. Abortive synthesis of trinucleotides in a dinucleotide‐primed reaction proceeded at roughly wild‐type levels, indicating that the mutation did not affect the formation of the first phosphodiester bond, but rather the transition from abortive initiation to processive elongation. In specific transcription assays, on the SUP4 tRNA gene, pausing was extended but the rate of RNA elongation between pause sites was not affected. Finally, the rate of cleavage of nascent RNA transcripts by halted mutant RNA polymerase was increased approximately 10‐fold. We propose that the domain f mutation affects the transition between two transcriptional modes, one being adopted during abortive transcription and at pause sites, the other during elongation between pause sites.

Low concentrations of the antibiotic doxycycline are sufficient to control the activity of an allosteric hammerhead ribozyme (shown schematically). This was obtained by allosteric selection of an RNA library, which was fused to helix II of the ribozyme. Such molecular switches may serve as a valuable tool for the development of tailored conditional gene expression systems that can be controlled by the presence or absence of any kind of small molecule.