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

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Abstract
The Human Immunodeficiency Virus type 1 Tat protein interacts specifically with a U-rich bulge within an RNA stem−loop known as the trans-activation 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 U23 in the bulge by 4-thioU. We have identified the cross-linked amino acid as Arg55 in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MALDI−TOF mass spectrometric analysis. The identification also required use of a synthetic Tat peptide containing a site-specific, uniformly 13C and 15N isotopically labeled arginine. We also describe a new chemical procedure for obtaining site-specific cross-links to Tat via the use of 2‘-β-alanyl U-substituted TAR and the amino-specific reagent dithiobis(succinimidyl propionate). U23-2‘-functionalized TAR was shown to cross-link uniquely to Lys51 in the basic region of Tat, whereas other sites in the upper and lower stems of TAR (U35, U38, and U42) showed cross-linking only to the N-terminus of Tat peptide (37−72). U40 cross-linked to both Lys51 and the N-terminus of the peptide. The results help to establish a preliminary model of the binding of Tat peptide to the major groove of TAR RNA.

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