Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region

R. Gaynor, E. Soultanakis, M. Kuwabara, J. Garcia, D. S. Sigman

Research output: Contribution to journalArticle

63 Scopus citations

Abstract

The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical 'imprinting' has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhance cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein.

Original languageEnglish (US)
Pages (from-to)4858-4862
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number13
DOIs
StatePublished - Jan 1 1989

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