Arginine-mediated RNA recognition: the arginine fork

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral gene expression.
• Tat protein interacts with the transactivation response (TAR) element of viral RNA.
• The basic region of Tat is known to bind TAR RNA, but the precise molecular interactions are not fully elucidated.

Purpose of the Study:

• To investigate the role of specific amino acid residues, particularly arginine, in the binding of Tat-derived peptides to TAR RNA.
• To determine the contribution of these interactions to the transactivation activity of the Tat protein.
• To elucidate the structural basis for arginine-mediated recognition of RNA bulges.

Main Methods:

• Peptide synthesis and binding assays using TAR RNA.
• Site-directed mutagenesis of the Tat protein.
• In vitro transactivation assays.
• Ethylation interference experiments.
• Molecular modeling and structural analysis.

Main Results:

• A peptide containing nine arginines (R9) showed specific binding to TAR RNA.
• A mutant Tat protein with R9 exhibited full transactivation activity.
• A peptide with nine lysines (K9) bound TAR poorly, with the corresponding protein showing marginal activity.
• Identification of a single arginine residue critical for specific binding and transactivation.
• Ethylation interference and modeling suggest arginine contacts adjacent phosphates in the RNA bulge.

Conclusions:

• Arginine side chains are critical for specific binding of Tat peptides to TAR RNA bulges.
• Arginine-mediated hydrogen bonding networks with RNA phosphates likely facilitate recognition of RNA structural motifs.
• Arginine may be a common residue utilized by proteins to recognize specific RNA structures, particularly loops and bulges.