Studies were performed to verify the physiological significance of attenuation in the life cycle of simian virus 40 and the role of agnoprotein in this process. For these purposes, nuclei were isolated at various times after infection and incubated in vitro in the presence of [a-32PJUTP under the standard conditions which lead to attenuation. Attenuation was evident by the production of a 94-nucleotide attenuator RNA, revealed by gel electrophoresis. In parallel, the synthesis of agnoprotein was studied at various times after infection by labeling the cells for 3 h with ["4C] The structural similarity between the major leader of 16S mRNA of simian virus 40 (SV40) and the leader of amino acid operons in bacteria is striking (3,14,40). This similarity raised the possibility that the major leader of 16S mRNA participates in an attenuation mechanism that regulates the expression of the downstream structural gene, VP1 (2,14). Like the leaders of amino acid operons in bacteria, the major leader of 16S mRNA can be folded into two alternative secondary structures. One of the structures consists of two stem-and-loop structures (1 and 2; 3 and 4) and was termed the attenuation conformation (Fig. 1A) (14). The alternative secondary structure contains one stem-and-loop structure (2 and 3) which overlaps with that of the attenuation conformation. This structure was termed the readthrough conformation ( Fig. 1B; 14). The 3 and 4 stem and loop, in the attenuation conformation, is followed by a run of uridylic acid residues. This structure acts as a transcription termination signal which prevents transcription from continuing into the structural gene (12, 32). In a further analogy to the leader sequences of the mRNAs from amino acid operons in bacteria, the major leader of 16S mRNA of SV40 codes for a small protein known as agnoprotein (13,16,17,26,27).In bacteria, where transcription and translation are coupled, the translation process of the leader peptide regulates the attenuation of transcription (40). We have suggested that in SV40, where transcription and translation are uncoupled, the final product of translation of the leader's codons, i.e., the agnoprotein, regulates the attenuation of transcription (2,3,14). According to this suggestion, the agnoprotein is transported from the cytoplasm to the nucleus and has a dual function. In the nucleus it enhances the attenuation of transcription by stabilizing the attenuation conformation in the nascent RNA (Fig. 1A). In the cytoplasm, by stabilizing * Corresponding author.the same conformation at the 5' end of the 16S mRNA, it enhances the translation of VP1. In this conformation the AUG start codon of the agnoprotein is sequestered in the 1;2 stem-and-loop structure (Fig. IA), and the scanning ribosome bypasses it (18-24) and initiates translation from the downstream AUG of VP1. A similar mechanism has been suggested to regulate the synthesis of VP2 and VP3 (2, 3). It has been postulated that attenuation during SV40 late transcription regulates the synthesis of the amounts of ...
