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.
Mucha
Piotr
P
Department of Chemistry, University of Gdansk, Poland. fly@chemik.chem.univ.gda.pl
Szyk
Agnieszka
A
Rekowski
Piotr
P
Barciszewski
Jan
J
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry; D015534:Trans-Activators / Q000032:analysis*; D015533:Transcriptional Activation
D012367:RNA, Viral; D015534:Trans-Activators; D001120:Arginine