Three distinct classes of S-adenosyl-l-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The SMK box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-Å crystal structure of the Enterococcus faecalis SMK box riboswitch. The Y-shaped riboswitch organizes its conserved nucleotides around a three-way junction for SAM recognition. The Shine-Dalgarno sequence, which is sequestered by base-pairing with the anti–Shine-Dalgarno sequence in response to SAM binding, also directly participates in SAM recognition. The riboswitch makes extensive interactions with the adenosine and sulfonium moieties of SAM but does not appear to recognize the tail of the methionine moiety. We captured a structural snapshot of the SMK box riboswitch sampling the near-cognate ligand S-adenosyl-l-homocysteine (SAH) in which SAH was found to adopt an alternative conformation and fails to make several key interactions.

S-box (SAM-I) riboswitches are a widespread class of riboswitches involved in the regulation of sulfur metabolism in Gram-positive bacteria. We report here the 3.0-Å crystal structure of the aptamer domain of the Bacillus subtilis yitJ S-box (SAM-I) riboswitch bound to S-adenosyl-L-methionine (SAM). The RNA folds into two sets of helical stacks spatially arranged by tertiary interactions including a K-turn and a pseudoknot at a four-way junction. The tertiary structure is further stabilized by metal coordination, extensive ribose zipper interactions, and SAM-mediated tertiary interactions. Despite structural differences in the peripheral regions, the SAM-binding core of the B. subtilis yitJ riboswitch is virtually superimposable with the previously determined Thermoanaerobacter tengcongensis yitJ riboswitch structure, suggesting that a highly conserved ligand-recognition mechanism is utilized by all S-box riboswitches. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing analysis further revealed that the alternative base-pairing element in the expression platform controls the conformational switching process. In the absence of SAM, the apo yitJ aptamer domain folds predominantly into a pre-binding conformation that resembles, but is not identical with, the SAM-bound state. We propose that SAM enters the ligand-binding site through the “J1/2–J3/4” gate and “locks” down the SAM-bound conformation through an induced-fit mechanism. Temperature-dependent SHAPE revealed that the tertiary interaction-stabilized SAM-binding core is extremely stable, likely due to the cooperative RNA folding behavior. Mutational studies revealed that certain modifications in the SAM-binding region result in loss of SAM binding and constitutive termination, which suggests that these mutations lock the RNA into a form that resembles the SAM-bound form in the absence of SAM.

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage ϕ29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular basepairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of ϕ29 DNA.D uring the assembly of the Bacillus subtilis bacteriophage ϕ29 and other double-stranded DNA phages, an ATP-driven ring motor plays a crucial role in packaging viral DNA to near crystalline density inside preformed protein shells (proheads) (1). In ϕ29, the packaging motor is comprised of three ring structures (Fig. 1A). The head-tail connector, a dodecameric ring of gp10, is embedded in the portal vertex of the head and provides a channel for entry and exit of the genome (2). An oligomeric ring of ϕ29 encoded prohead RNA (pRNA) encircles the protruding end of the connector, displaying five "spokes" that project away from the head (2, 3). Subunits of the viral packaging ATPase gp16, a member of the FtsK/HerA ring motor family (4), form a ring that contacts the five spokes of pRNA, completing the packaging motor (3, 5, 6). Single-molecule laser tweezers studies showed that the ϕ29 DNA packaging motor is one of the strongest molecular motors known, generating approximately 65 pN force (compared to approximately 3 pN for muscle myosin) (7). The requirement of a RNA-ring structure in the assembly and function of the ϕ29 DNA packaging motor contrasts with equivalent motors in other dsDNA phages such as T4, SPP1, lambda, and P22 where protein-protein contacts anchor ring ATPases to the phage proheads (1). Cryoelectron microscopy (cryo-EM) 3D reconstructions (2,3,8) show that pRNA is strategically positioned in the ϕ29 packaging motor to link the capsid, connector and ATPase components of the motor (Fig. 1A).Besides being an essential component of the motor, the ϕ29 pRNA multimer is a rare example of a self-assembling RNA that functions at the quaternary...

The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N(6)-cyclohexyladenine 2] was found to be a moderately potent antagonist for the human A(3) adenosine receptor (AR) with a K(i) value of 0.66 microM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-(phenylamino)-N(6)-cyclohexyladenine (12), 2-(phenylamino)-N(6)-cycloheptyladenine (19), and 2-phenylamino-N(6)-endo-norbornyladenine (21) as potent A(3) AR ligands with K(i) values of 51, 42, and 37 nM, respectively, with 30-200-fold selectivity in comparison to A(1) and A(2A) ARs. The most selective A(3) AR antagonist (>200-fold) was 2-(phenyloxy)-N(6)-cyclohexyladenine (22). 9-Methylation of 12, but not 19, was well-tolerated in A(3) AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-(phenylamino), 2-(phenyloxy), and 2-(phenylthio) substitutions, the order of affinity at the A(3) AR was oxy > or = amino > thio. Selected derivatives, including reversine (K(B) value of 466 nM via Schild analysis), competitively antagonized the functional effects of a selective A(3) AR agonist, i.e., inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N(6) position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A(3) AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A(3) AR antagonist effects and dedifferentiation.