The 5e motif of eukaryotic signal recognition particle RNA contains a conserved adenosine for the binding of SRP72

Abstract

The signal recognition particle (SRP) plays a pivotal role in transporting proteins to cell membranes. In higher eukaryotes, SRP consists of an RNA molecule and six proteins. The largest of the SRP proteins, SRP72, was found previously to bind to the SRP RNA. A fragment of human SRP72 (72c9) bound effectively to human SRP RNA but only weakly to the similar SRP RNA of the archaeon Methanococcus jannaschii. Chimeras between the human and M. jannaschii SRP RNAs were constructed and used as substrates for 72c9. SRP RNA helical section 5e contained the 72c9 binding site. Systematic alteration within 5e revealed that the A240G and A240C changes dramatically reduced the binding of 72c9. Human SRP RNA with a single A240G change was unable to form a complex with full-length human SRP72. Two small RNA fragments, one composed of helical section 5ef, the other of section 5e, competed equally well for the binding of 72c9, demonstrating that no other regions of the SRPR RNA were required. The biochemical data completely agreed with the nucleotide conservation pattern observed across the phylogenetic spectrum. Thus, most eukaryotic SRP RNAs are likely to require for function an adenosine within their 5e motifs. The human 5ef RNA was remarkably resistant to ribonucleolytic attack suggesting that the 240-AUC-242 ''loop'' and its surrounding nucleotides form a peculiar compact structure recognized only by SRP72.

Characterization of the SRP68/72 interface of human signal recognition particle by systematic site‐directed mutagenesis

“…21 Lysates containing polypeptide 68f (residues 562 to 620) expressed from a Kanamycin resistant plasmid (''Materials and Methods'' section) were mixed with 6xHis-tagged 72b 0 (H72b 0 ) and samples were analyzed using the assay depicted in Figure 3 The N-terminally 6xHis-tagged 68h peptide (H68h) contained the 39 amino acid residues at positions 570 to 605 and was encoded by a Kanamycin resistance plasmid. Different E. coli host strains and growth conditions were explored to express H68h, but all attempts to isolate intact H68h proved unsuccessful.…”

“…21 Plasmids p68e 0 and p68f for the expression of untagged polypeptides 68e 0 (residues 530 to 620) and 68f (562 to 620) respectively were generated by PCR from the pThx-H68e 0 followed by cloning of the amplified restricted DNAs into Kanamycin resistant pET28a (Novagen). His-tagged 68h (H68h, residues 570 to 605) was assembled by ligation of two complementary synthetic oligonucleotides compatible with the NcoI and EcoRI restrictions sites of pET28a.…”

“…We have shown that a single conserved adenosine residue within the eukaryotic 5e motif is essential for the formation of a complex with human SRP72. 21 This portion of the SRP RNA binds to a relatively small 56-residue region near the C-terminus of SRP72 into which the consensus sequence PDPXRWLPXXER is imbedded. In contrast, the expansive RNA binding region of human SRP68 engages amino acid residues from positions 52 to 252.…”

Kinship in the SRP RNA family

“…1A). 36 In archaea, which lack protein SRP72, the 5e motif may contribute to the formation of an interdomain hinge or kink as observed by cryo-electron microscopy of the elongation-arrested ribosome bound canine SRP. 37,38 In contrast to the detailed structural information of complexes containing SRP9/14, SRP19 and SRP54, high-resolution data about SRP68/72 are unavailable.…”

“…This adenosine (A240 in human SRP RNA) was found to be essential for the binding of protein SRP72. 36 The UGU(NR) motif (abbreviated as UGU in Fig. 1A) of the small domain, either connected helices 3 and 4 or was part of the helix 2 hairpin in the fungal SRP RNAs (Fig.…”