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To characterize the transcriptional organization and regulation of minute virus of mice, an autonomous parvovirus, viral transcriptional complexes were isolated and cleaved with restriction enzymes. The in vivo preinitiated nascent RNA was elongated in vitro in the presence of [ot-32P]UTP to generate runoff transcripts. The lengths of the runoff transcripts were analyzed by gel electrophoresis under denaturing conditions. On the basis of the map locations of the restriction sites and the lengths of the runoff transcripts, the in vivo initiation sites were determined. Two major initiation sites having similar activities were thus identified at residues 201 ± 5 and 2005 ± 5; both of them were preceded by a TATAA sequence. When uncleaved viral transcriptional complexes or isolated nuclei were incubated in vitro in the presence of [a-32P]UTP or [0t-32P]CTP, they synthesized labeled RNA that, as determined by polyacrylamide gel electrophoresis, contained a major band of 142 nucleotides. The RNA of the major band was mapped between the initiation site at residue 201 ± 5 and residue 342. We noticed the potential of forming two mutually exclusive stem-and-loop structures in the 142nucleotide RNA; one of them is followed by a string of uridylic acid residues typical of a procaryotic transcription termination signal. We propose that, as in the transcription of simian virus 40, RNA transcription in minute virus of mice may be regulated by attenuation and may involve eucaryotic polymerase B, which can respond to a transcription termination signal similar to that of the procaryotic polymerase.
To characterize the transcriptional organization and regulation of minute virus of mice, an autonomous parvovirus, viral transcriptional complexes were isolated and cleaved with restriction enzymes. The in vivo preinitiated nascent RNA was elongated in vitro in the presence of [ot-32P]UTP to generate runoff transcripts. The lengths of the runoff transcripts were analyzed by gel electrophoresis under denaturing conditions. On the basis of the map locations of the restriction sites and the lengths of the runoff transcripts, the in vivo initiation sites were determined. Two major initiation sites having similar activities were thus identified at residues 201 ± 5 and 2005 ± 5; both of them were preceded by a TATAA sequence. When uncleaved viral transcriptional complexes or isolated nuclei were incubated in vitro in the presence of [a-32P]UTP or [0t-32P]CTP, they synthesized labeled RNA that, as determined by polyacrylamide gel electrophoresis, contained a major band of 142 nucleotides. The RNA of the major band was mapped between the initiation site at residue 201 ± 5 and residue 342. We noticed the potential of forming two mutually exclusive stem-and-loop structures in the 142nucleotide RNA; one of them is followed by a string of uridylic acid residues typical of a procaryotic transcription termination signal. We propose that, as in the transcription of simian virus 40, RNA transcription in minute virus of mice may be regulated by attenuation and may involve eucaryotic polymerase B, which can respond to a transcription termination signal similar to that of the procaryotic polymerase.
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 ...
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 [alpha-32P]UTP 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 [14C]arginine, lysing them, and analyzing the labeled proteins by gel electrophoresis. Both attenuation and the synthesis of agnoprotein were predominant towards the end of the infectious cycle. At earlier times, there was almost no attenuation and no synthesis of agnoprotein. Moreover, there was almost no attenuation even at the latest times after infection in nuclei isolated from cells infected with simian virus 40 deletion mutants that do not synthesize agnoprotein. Finally, analysis by dot blot hybridization showed higher amounts of cytoplasmic viral RNA in cells infected with an agnoprotein gene insertion mutant, delta 79, that does not produce agnoprotein, compared with cells infected with wild-type virus. The present studies indicate that attenuation is temporally regulated and suggest that agnoprotein enhances attenuation in isolated nuclei and that may also enhance it in vivo.
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