Transcriptional control signals of a model eukaryotic protein-coding gene have been identified by a new procedure of in vitro mutagenesis. This method allows small clusters of nucleotide residues to be substituted in a site-directed manner without causing the addition or deletion of other sequences. Transcription assays of a systematic series of these clustered point mutants have led to the identification of three distinct control signals located within the 105-nucleotide residues immediately upstream from the point where transcription begins.

Mature virions of herpes simplex virus type 1 contain an activating factor that primes transcription from the five virally encoded immediate early (IE) genes. This activator is specified by a 65-kD polypeptide termed VP16. The action of VP16 is mediated through cis-regulatory elements located in regions adjacent to each IE gene. Although VP16 is normally introduced into cells by infecting virions, its trans-activating function can also be observed by cotransfecting cells with a plasmid that encodes VP16 along with a reporter gene driven by IE cisregulatory sequences. We have used such an assay to examine the function of mutant forms of VP16. Our results provide tentative identification of two domains of VP16 that are crucial to its role in the induction of IE gene expression. One domain is located within the carboxy-terminal 78 amino acids of VP16 and is characterized by its acidity. Another domain, located in a more amino-terminal region of the protein, appears to tailor the specificity of VP16 for IE genes. According to the results presented in this and the accompanying paper, we predict that VP16 achieves IE gene specificity via protein : protein, rather than protein : DNA, interaction.

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The two distal transcription signals of the herpesvirus tk gene share a common hexanucleotide sequence and function in a mutually dependent manner. We examined their roles by introducing corresponding base mutations into each hexanucleotide. The effects of mutations in the hexanucleotide of the first distal signal match the pattern of effects of the corresponding mutations in the second distal signal. These concordant patterns suggest that the hexanucleotide repeats are functionally analogous. There is one difference between the two distal signals. Disruption of the second distal signal concomitantly inactivates the first distal signal; disruption of the first distal signal leads to only partial inactivation of the second distal signal. When the positions of the two distal signals are switched, the partial dependence of the second distal signal is alleviated. We suggest a model for positive transcriptional control analogous to activation of the cl maintenance promoter of bacteriophage lambda by repressor.