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 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'-beta-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.
Farrow
M A
MA
Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, U.K.
Aboul-ela
F
F
Owen
D
D
Karpeisky
A
A
Beigelman
L
L
Gait
M J
MJ
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000378:metabolism*; D015497:HIV-1; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013891:Thiouridine / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D000345:Affinity Labels; D000596:Amino Acids; D003432:Cross-Linking Reagents; D015696:Gene Products, tat; D010446:Peptide Fragments; C111139:Tat peptide (37-72), Human immunodeficiency virus 1; D054322:tat Gene Products, Human Immunodeficiency Virus; D013891:Thiouridine; D001120:Arginine