Arginine-binding RNAs resembling TAR identified by in vitro selection

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The human immunodeficiency virus (HIV) Tat protein binds to its RNA target (TAR) primarily through an arginine residue.
• A proposed NMR model describes arginine binding to a guanine base and phosphates within the TAR RNA structure.

Purpose of the Study:

• To identify and characterize other RNA structures that bind arginine with similar affinity to TAR.
• To investigate the structural versatility of arginine-binding sites in RNA and how surrounding sequences modulate binding.

Main Methods:

• In vitro selection experiments to identify high-affinity arginine-binding RNAs.
• Chemical modification and RNase mapping to probe RNA structure and nucleotide involvement in binding.
• Measurement of arginine-binding affinities for various RNA constructs.

Main Results:

• Approximately 40% of selected RNAs shared a conserved motif with TAR, featuring two stems separated by a bulge and specific base pairing.
• "TAR-like" RNAs demonstrated similar arginine-binding characteristics to TAR, with some binding influenced by nucleotides outside the primary site.
• A UAG loop in TAR-like RNAs enhanced arginine binding affinity, inducing a conformational change from an unstructured loop to a bulge.

Conclusions:

• The arginine-binding site in TAR is structurally adaptable.
• Surrounding RNA sequences and loop structures significantly influence and modulate arginine binding affinity and mechanism.