RNA recognition and base flipping by the toxin sarcin

Abstract

Sarcin is a member of a fungal toxin family that enters cells and specifically cleaves one of the thousands of RNA phosphodiester bonds in the ribosome. As a result, elongation factor binding is disrupted, translation is inhibited and apoptosis is triggered. The toxin targets a universal RNA structure in the ribosome called the sarcin/ricin loop (SRL). A 1.11 Å resolution structure of a minimal SRL RNA substrate (∼30‐mer) shows that the loop portion of the substrate folds into two common building blocks of RNA structure: a bulged‐G motif (recognition site) and a GAGA tetraloop (cleavage site). To elucidate the structural basis of toxin action, two co‐crystal structures of the sarcin homologue restrictocin have beeen determined bound to different analogs of a minimal SRL RNA substrate. Our studies argue that site selection by the toxin depends on direct base and shape recognition of the SRL RNA, and that cleavage by the toxin depends on a base flipping mechanism that positions the nucleophile for in‐line attack on the scissile bond.

The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from Its Structure in Crystals of 50S Ribosomal Subunits,

“…the initial structure may serve as a "book mark" identifying the site to be opened. Interestingly, Restrictocin-a sarcin like toxin recognizes a bulged-G type S-turn in the sarcin/ricin tetraloop of rat 23S rRNA and flips a G on the 5' side of the cleavage site facilitating in-line orientation of the 2'OH of G with the P-O5 of the 3' A (82,83). The nearest neighbor model (2, 3) predicts a free energy increment of 3.53 kcal/mol (Table 2) for the bulged internal loop structure found in crystals of the LSU of D. radiodurans (4) and E. coli (5).…”

Modeling the highly specific ribotoxin recognition of ribosomes

“…It seems clear now that after their action the SRL and the ribosomal components in its vicinity become more rigid, impairing the conformational changes needed for the essential GTP hydrolysis exerted by the elongation factors [24]. However, the structural bases to explain their exquisite action are not yet completely understood despite having been thoroughly studied [25][26][27].…”

Assessment of adenyl residue reactivity within model nucleic acids by surface enhanced Raman spectroscopy

“…As for A tracts in (pA) n and ss d9-mer, concerted A loading could lower the energetic barrier for A unstacking and backbone twisting. 42 Based on presently ranked A reactivity and previous studies, 6,[22][23][24][25][26][27][74][75][76][77]114 recognition of rGNRA loops are certainly related to their structure. A docking finds different routes according to initial availability.…”

“…The silver sites used to rate A reactivity are crudely representative of electropositive domains surrounding ribosomal 23,24 and viral proteins. 21 For example, a ribosomal sarcin homologue enzyme binds a base-flipped form of a rGAGA loop.…”

“…20,21 A great deal of attention has been focused on thermodynamic, structural, and biological properties of the highly conserved ribo polynucleotide (r)GNRA tetraloops. 6,15,17,[21][22][23] While X-ray studies show that these loops share common structural features, 24 dynamic simulations, 25 vibrational, 15 UV, 17 and NMR 26,27 analyses conjointly provide evidence that local dynamics, hydrogen bonding, and base stacking depend on primary sequences. Double strand pairing dominates in DNA, but the discovery of inverted repeats (palindromes) led to the revelation that deoxy ribo polynucleotide (d) hairpin structures (cruciforms) promote biological activity.…”