Crystal structure of the ribosomal RNA domain essential for binding elongation factors

Abstract

The structure of a 29-nucleotide RNA containing the sarcin͞ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be f lexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the ''f lexible'' region and to a lesser extent in the G-bulged cross-strand A stack.The highly conserved sarcin͞ricin loop (SRL), a component of 23-28 S rRNA, is essential for protein synthesis because it participates in the binding of elongation factors (EFs) to the ribosome. The function of this domain was illucidated by studying ribosomes treated with its namesake ribotoxins, sarcin and ricin (1, 2). The ribotoxins cleave a single covalent bond in the SRL RNA, which kill cells by inactivating ribosomes. Sarcin is a ribonuclease that cleaves the phosphodiester backbone on the 3Ј-side of G4325 (rat 28 S rRNA numbering is used throughout) (1, 2). Ricin depurinates the 5Ј-adjacent A4324 (3). EF-dependent functions are specifically impaired when ribosomes are treated with either toxin, whereas other ribosomal functions including EF-G-independent translocation are unaffected (1). Further indications of the domain's importance are that 12 of its 17 nucleotides are near universal and that chemical footprinting (4) marks it as the only rRNA region protected by both EFs.An SRL RNA can mimic the SRL in the ribosome because binding of EF-G to an Escherichia coli or rat SRL RNA is only Ϸ10-fold weaker than binding to intact E. coli ribosomes (5). Further, only the GTP-bound and apoprotein forms of EF-G bind the E. coli SRL RNA, whereas GDP-bound EF-G does not (5). EF-G binding is most affected by mutation of G4319 (5). Both toxins recognize and cleave a single bond in the SRL oligonucleotide (Fig. 1). Comprehensive mutational analysis of the SRL established that sarcin recognition and cleavage are largely dependent on a single base, the bulged G4319 (7). In contrast, each of the 4 nt of the GAGA tetraloop is necessary, and a GAGA tetraloop is sufficient for ricin recognition and depurination (6).The perception of RNA loop structure was altered by the determination of the conformation of the rat SRL by NMR spectroscopy (8, 9). Because the rules that govern formation of non-Watson-Crick ...

Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

“…The SRL tetraloop assumes different conformations before and after binding restrictocin (11,16) (Fig. S2).…”

“…If so, what explains the significant difference in k cat /K m between these two substrates [10 6 vs. 10 4 M Ϫ1 s Ϫ1 at ionic strength Ϸ100 mM (1)]? (ii) Comparison of the SRL RNA structure bound to restrictocin with the unbound counterpart (16) shows that the tetraloop, consisting of G4323-A4326, becomes unfolded and the bases flip out (Fig. S2).…”

Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

“…The biological activity of recombinant PAP was virtually identical to that of native PAP purified from the pokeweed plant (22). The availability of recombinant PAP provides a unique opportunity for structure-activity relationship (SAR) analyses.…”

“…We have recently reported the expression of biologically active recombinant PAP in Escherichia coli (22). The biological activity of recombinant PAP was virtually identical to that of native PAP purified from the pokeweed plant (22).…”

“…Ϫ expression vector (22). Uracil-containing template of PAP was obtained by transforming E. coli CJ236 with the recombinant plasmid phosphatebuffered saline-PAP.…”