Physical Mapping of HIV Reverse Transcriptase to the 5′ End of RNA Primers

• Published in: The Journal of biological chemistry Vol. 276; no. 35; pp. 32515 - 32521
• Main Authors: DeStefano, Jeffrey J., Cristofaro, Jason V., Derebail, Suchitra, Bohlayer, William P., Fitzgerald-Heath, Megan J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31.08.2001
• Subjects: 3' Untranslated Regions - chemistry; 3' Untranslated Regions - metabolism; 5' Untranslated Regions - chemistry; 5' Untranslated Regions - metabolism; Amino Acid Substitution; Base Sequence; Binding Sites; Escherichia coli - enzymology; HIV Reverse Transcriptase - chemistry; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; HIV-1 - enzymology; Human immunodeficiency virus; Humans; Kinetics; Molecular Sequence Data; Nucleic Acid Hybridization; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Ribonuclease H - genetics; Ribonuclease H - metabolism; Substrate Specificity; Templates, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M103958200

Enzymatic analysis of RNA cleavage products has suggested that human immunodeficiency virus (HIV) reverse transcriptase (RT) binds to the 5′ end of RNAs that are recessed on a longer DNA template (RNA primers) yet binds to the 3′ end of DNA primers. One concern is that RT molecules bound at the 3′ end of RNA would not be easily detected because RT may not catalyze substantial RNA extension or cleavage when bound to the 3′ end. We used physical mapping to show that RT binds preferentially to the 5′ end of RNA primers. An HIV-RT that lacked RNase H activity (HIV-RTE478Q) was incubated with the RNA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT protected a ∼23-base region at the 5′ end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal structure of HIV-RT. No protection of the RNA 3′ end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wild-type HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.