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

被引:28
作者
Farrow, MA
Aboul-ela, F
Owen, D
Karpeisky, A
Beigelman, L
Gait, MJ
机构
[1] MRC, Mol Biol Lab, Cambridge CB2 2QH, England
[2] Ribozymer Pharmaceut Inc, Dept Chem & Biochem, Boulder, CO 80301 USA
关键词
D O I
10.1021/bi972695v
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 U-23 in the bulge by 4-thioU. We have identified 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 MALDI-TOF mass spectrometric analysis. The identification also required use of a synthetic Tat peptide containing a site-specific, uniformly C-13 and N-15 isotopically labeled arginine. We also describe a new chemical 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 shown to cross-link uniquely to Lys(51) in the basic region of Tat, whereas 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 Tat peptide to the major groove of TAR RNA.
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页码:3096 / 3108
页数:13
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