Identification of amino acids that promote specific and rigid TAR RNA-tat protein complex formation.
The Tat protein and the transactivation responsive (TAR) RNA form an essential complex in the HIV lifecycle, and mutations in the basic region of the Tat protein alter this RNA-protein molecular recognition. Here, EPR spectroscopy was used to identify amino acids, flanking an essential arginine of the Tat protein, which contribute to specific and rigid TAR-Tat complex formation by monitoring changes in the mobility of nitroxide spin-labeled TAR RNA nucleotides upon binding. Arginine to lysine N-terminal mutations did not affect TAR RNA interfacial dynamics. In contrast, C-terminal point mutations, R56 in particular, affected the mobility of nucleotides U23 and U38, which are involved in a base-triple interaction in the complex. This report highlights the role of dynamics in specific molecular complex formation and demonstrates the ability of EPR spectroscopy to study interfacial dynamics of macromolecular complexes.
Edwards
Thomas E
TE
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Robinson
Bruce H
BH
Sigurdsson
Snorri Th
ST
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1; D008762:Methylhistidines; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D013816:Thermodynamics; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
D000596:Amino Acids; D015696:Gene Products, tat; D008762:Methylhistidines; D046912:Multiprotein Complexes; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein; C061475:ovothiol A