The conserved adenosine in helix 6 of Archaeoglobus fulgidus signal recognition particle RNA initiates SRP assembly
Jiaming Yin (1), Qiaojia Huang (1, 2), Olga N. Pakhomova (3), Andrew P. Hinck (3) and Christian Zwieb (1, 4)
1. Department of Molecular Biology, University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708-3154, USA / 2. Department of Laboratory Medicine, Fuzhou General Hospital, 156 North Xihuan Road, Fuzhou 350025, Fujian, P.R. China / 3. Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA / 4. Corresponding author ([email protected]) / Received December 5, 2003; accepted April 26, 2004; published online June 11, 2004
The signal recognition particle (SRP) RNA helix 6 of archaea and eukaryotes is essential for the binding of protein SRP19 and the assembly of a functional complex. The conserved adenosine at the third position of the tetraloop of helix 6 (A149) is crucial for the binding of protein SRP19 in the mammalian SRP. Here we investigated the significance of the equivalent adenosine residue at position 159 (A159) of Archaeoglobus fulgidus SRP RNA. The A159 of A. fulgidus and A149 of human SRP RNA were changed to C, G or U, and fragments containing helix 6 or helices 6 and 8 were synthesized by run-off transcription with T7 RNA polymerase. The ability of recombinant A. fulgidus and human SRP19 to form ribonucleoprotein complexes was measured in vitro. The simultaneous presence of A149 and helix 8 is required for the high-affinity binding of SRP19 to the human SRP RNA. In contrast, A. fulgidus SRP19 binds to the SRP RNA fragments with high affinity irrespective of the nature of the nucleotide, demonstrating that A159 does not directly participate in protein binding. Instead, as indicated by the resistance of the wild-type A. fulgidus RNA towards digestion by RNase A, this residue allows the formation of a tightly folded RNA molecule. The high affinity between A. fulgidus SRP19 and RNA molecules that contain both helices 6 and 8 suggests that A159 is likely to initiate archaeal SRP assembly by forming a conserved tertiary RNA– RNA interaction.
Keywords: protein–RNA interactions, site-directed mutagenesis, tetraloop.