SITE-SPECIFIC CROSS-LINKING OF AMINO-ACIDS IN THE BASIC REGION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT PEPTIDE TO CHEMICALLY-MODIFIED TAR RNA DUPLEXES

The Human Immunodeficiency Virus type 1 Tat protein interacts specific ally with a U-rich bulge within an RNA stem-loop known as the trans-ac tivation responsive region (TAR) that occurs in all viral transcripts. We have photochemically cross-linked to Tat peptide (37-72), a model TAR duplex substituted at U-23 in the bulge by 4-thioU. We have identi fied the cross-linked amino acid as Arg(55) in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MAL DI-TOF mass spectrometric analysis. The identification also required u se of a synthetic Tat peptide containing a site-specific, uniformly C- 13 and N-15 isotopically labeled arginine. We also describe a new chem ical procedure for obtaining site-specific cross-links to Tat via the use of 2'-beta-alanyl U-substituted TAR and the amino-specific reagent dithiobis(succinimidyl propionate). U-23-2'-functionalized TAR was sh own to cross-link uniquely to Lys(51) in the basic region of Tat, wher eas other sites in the upper and lower stems of TAR (U-35, U-38, and U -42) showed cross-linking only to the N-terminus of Tat peptide (37-72 ). U-40 crosslinked to both Lys(51) and the N-terminus of the peptide. The results help to establish a preliminary model of the binding of T at peptide to the major groove of TAR RNA.