Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation responsive element with trans-activator of transcription protein (49–57): Capillary electrophoresis mobility shift assay

A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested that a single, conserved arginine residue (presumably Arg52) within the arginine-rich region (ARR) of Tat plays the major role for the Tat–TAR recognition. To study structural requirements of the Arg52 position, Tat(49–57)–NH2 analogues substituted with nonencoded amino acids at the Arg52 position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49–57) a significant shift of migration time of TAR from 18.66 min (RSD=1.4%) to 20.12 min (RSD=2.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg52 [L-Arg52→Cit, →Orn, →Arg(NO2), →Arg(Me2)] strongly disrupted or abolished the TAR–Tat peptide interaction. Enantiomeric substitution, L-Arg52→D-Arg was the only one which notably promoted TAR–Tat peptide interaction. The results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg52 plays a crucial role for TAR–Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA–peptide complex formation. The methodology presented should be generally applicable to study RNA–peptide (protein) interaction.