PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during biosynthesis of the essential tRNA base queuosine. We report the first preQ1-II riboswitch structure at 2.3 Å resolution, which uses a novel fold to achieve effector recognition at the confluence of a three-way-helical junction flanking a pseudoknotted ribosome-binding site (RBS). The results account for preQ1-II-riboswitch-mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory RNAs.

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18S ribosomal RNA in Xenopus laevis is 1,825 nucleotides long, as inferred from sequence analysis of an 18S gene. All the 40 rRNA methyl groups can be located in the sequence. Comparison with the yeast (Saccharomyces cerevisiae) 18S sequence reveals extensive regions of high homology interspersed with tracts having little or no homology. Regions of high homology contain almost all the RNa methyl groups. Major regions of low homology area considerably richer in C + G in Xenopus than in yeast.

. ) formed by menadione is attenuated, whereas induction by heme is not affected. We propose a role for BVR in the signaling cascade for AP-1 complex activation necessary for HO-1 oxidative stress response.

Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity. An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein. Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated. Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent. In addition, 2(3)-O-(2,4,6-trinitrophenyl)adenosine 5-triphosphate fluorescence titration of BVR gave a lower K d at pH 8.7 than at pH 7.4 (15.5 versus 28.0 M). Mn 2؉ was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60°C for 10 min. The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase. Potato acid phosphatase treatment reversibly inactivated the enzyme. The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition. Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate. The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H 2 O 2 . The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.

PreQ 1 -III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ 1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HL out -type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ 1 -III riboswitch aptamer forms a HL out -type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ 1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ 1 binds tightly (K D,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3′ RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ 1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (k dock ∼0.6 s −1 ) and undocking (k undock ∼1.1 s −1 ). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs. preQ 1 riboswitch | gene regulation | crystal structure | single-molecule FRET | molecular dynamics R iboswitches are structured RNA motifs that sense the cellular levels of small molecules to provide feedback regulation of genes (1). Although present in all domains of life, they are prominent in bacteria where they typically reside in the 5′-leader sequences of mRNA (2). Broad interest in riboswitches originates from the discovery that they can be targeted by antimicrobials (3-5), and the observation that they use complex scaffolds to achieve gene regulation without the need for protein partners. In the latter respect, riboswitches typically exhibit bipartite sequence organization comprising a conserved aptamer linked to a downstream expression platform (2). Aptamer binding to a cognate effector can induce conformational changes that alter the accessibility of expression platform sequences, such as those required for transcriptional read-through, or hybridization to the 16S rRNA as a preface to translation (2, 6).Numerous riboswitches fold as pseudoknots that conform to the H-type or closely related HL out -type topology, which have emerged as the most efficient RNA scaffolds to integrate aptamer and expression platform sequences (7). The preQ 1 -I, preQ 1 -II, S-adenosyl-L-methionine-II (SAM-II), and fluoride riboswitches are representative of this organizational strategy, and their analysis has contributed to a renaissance in our understanding of regulatory pseudoknot structure and dynamics (8-18). By contrast, pseudoknotted aptamers that do not integr...

This first, phase II, randomized study of pelareorep and paclitaxel in previously treated mBC did not show a difference in PFS (the primary endpoint) or RR. However, there was a significantly longer OS for the combination. Further exploration of this regimen in mBC may be of interest.

The methylated nucleotide sequences in the rRNA molecules of the following vertebrate cultured cells were compared: human (HeLa); hamster (BHK/C13); mouse (L); chick-embryo fibroblast; Xenopus laevis kidney. In each species the combined 18S, 28S and 5.8S molecules possess approx. 110-115 methyl groups, and the methylated oligonucleotides released after complete digestion of the rRNA by T1 ribonuclease encompass several hundred nucleotides. "Fingerprints" of the three mammalian methyl-labelled 18S rRNA species were qualitatively indistinguishable. "Fingerprints" of digests of 28S rRNA of hamster and mouse L-cells were extremely similar to those of HeLa cells, differing in one and three methylated oligonucleotides respectively. "Fingerprints" of methyl-labelled rRNA from chick and Xenopus strongly resembled those of mammals in most respects, but differed in several oligonucleotides in both 18S and 28S rRNA. At least some of the differences between "fingerprints" appear to be due to single base changes or to the presence or absence of methyl groups at particular points in the primary sequence. The findings strongly suggest that the methylated-nucleotide sequences are at least 95% homologous between the rRNA molecules of the two most distantly related vertebrates compared, man and Xenopus laevis.