RNA secondary structure is an integral part of the in vitro mechanism of attenuation in simian virus 40

• Published in: The Journal of biological chemistry 1989-06, Vol.264 (17), p.9953-9959
• Main Authors: Resnekov, O, Kessler, M, Aloni, Y
• Format: Article
• Language: English
• Subjects: Base Sequence; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Genes, Viral; HeLa Cells - metabolism; Humans; Kinetics; Models, Molecular; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Promoter Regions, Genetic; RNA, Viral - genetics; RNA, Viral - ultrastructure; Simian virus 40 - genetics; Templates, Genetic; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)81752-5

At late times after infection with SV40, a prematurely terminated transcript that initiates at the major late promoter (MLP) and has a 3′-end about 95 nucleotides downstream has been identified and termed an attenuated RNA (Hay, N., Skolnik-David, H., and Aloni, Y. (1982) Cell 29, 183–193). The DNA template of the attenuated RNA has two regions of dyad symmetry, and the attenuated RNA can therefore fold into two hairpin elements. The hairpin element at the 3′-end of the attenuated RNA is followed by a stretch of Us and resembles a ρ-independent terminator in prokaryotes. We have suggested that folding of the RNA into two hairpin elements will lead to a block of transcription elongation. Using site-directed mutagenesis, we created two templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared between the templates in a cell-free extract. We have shown that RNA polymerase II recognizes the SV40 sequence that leads to a block of transcription elongation, even when it is under the control of the MLP of adenovirus 2. The extent of the block of transcription elongation is directly dependent on the stability of the hairpin structure of the RNA as assessed by a comparison of transcription of the wild-type and mutated templates. The addition of Sarkosyl and transcription at an elevated temperature during the elongation reaction enhanced the production of the attenuated RNA from all templates.