Pascale Legault

NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site.

Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme.

NMR structure of the complex between the Tfb1 subunit of TFIIH and the activation domain of VP16: structural similarities between VP16 and p53.

The Herpes Simplex Virion Protein 16 (VP16) activates transcription through a series of protein/protein interactions involving its highly acidic transactivation domain (TAD). The acidic TAD of VP16 (VP16TAD) has been shown to interact with several partner proteins both in vitro and in vivo, and many of these VP16 partners also bind the acidic TAD of the mammalian tumor suppressor protein p53. For example, the TADs of VP16 and p53 (p53TAD) both interact directly with the p62/Tfb1 (human/yeast) subunit of TFIIH, and this interaction correlates with their ability to activate both the initiation and elongation phase of transcription. In this manuscript, we use NMR spectroscopy, isothermal titration calorimetery (ITC) and site-directed mutagenesis studies to characterize the interaction between the VP16TAD and Tfb1. We identify a region within the carboxyl-terminal subdomain of the VP16TAD (VP16C) that has sequence similarity with p53TAD2 and binds Tfb1 with nanomolar affinity. We determine an NMR structure of a Tfb1/VP16C complex, which represents the first high-resolution structure of the VP16TAD in complex with a target protein. The structure demonstrates that like p53TAD2, VP16C forms a 9-residue alpha-helix in complex with Tfb1. Comparison of the VP16/Tfb1and p53/Tfb1 structures clearly demonstrates how the viral activator VP16C and p53TAD2 shares numerous aspects of binding to Tfb1. Despite the similarities, important differences are observed between the p53TAD2/Tfb1 and VP16C/Tfb1 complexes, and these differences demonstrate how selected activators such as p53 depend on phosphorylation events to selectively regulate transcription.

p53 and TFIIEα share a common binding site on the Tfb1/p62 subunit of TFIIH

The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the α-subunit of the general transcription factor IIE (TFIIEα). We have determined that the acidic carboxyl terminus of TFIIEα (TFIIEα336–439) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEα binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEα336–439 is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEα336–439 and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEα336–439 contains a small domain (residues 395–433) folded in a novel ββααα topology. NMR mapping studies demonstrate that two unstructured regions (residues 377–393 and residues 433–439) located on either side of the folded domain appear to be required for TFIIEα336–439 binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEα336–439 can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEα and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.

NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1

FCP1 [transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase] is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II). The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans). It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436–517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879–961 in human). In this manuscript, we have determined a high-resolution solution structure of a cterRAP74/cterFCP complex by NMR spectroscopy. Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an α-helix (H1′; E945–M961) in the complex. The cterRAP74/cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1′ helix of cterFCP. The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1′ of cterFCP and lysine residues from both H2 and H3 of cterRAP74. There are also three additional polar interactions involving highly conserved acidic residues from the H1′ helix. The cterRAP74/cterFCP complex is the first high-resolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor. The structure defines a clear role for both hydrophobic and acidic residues in protein/protein complexes involving acidic residue-rich domains in transcription regulatory proteins.

Improved measurement of 13C, 31P J coupling constants in isotopically labeled RNA

3JCP coupling constants have been measured in 1 99% 13C, 15N labeled lead‐dependent ribozyme, known as the leadzyme. These coupling constants were determined by analysis of the intensity of individual crosspeaks in a spin‐echo difference constant time HSQC experiment. This procedure permits improved measurement of the 3JC2′P and 3JC4′P coupling constants in isotopically labeled RNA and yielded valuable information on the β and ϵ backbone torsion angles in the leadzyme.

Riboswitch structure: an internal residue mimicking the purine ligand

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson–Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39–C65 and A39–U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

Functional and structural characterization of a dense core secretory granule sorting domain from the PC1/3 protease

Several peptide hormones are initially synthesized as inactive precursors. It is only on entry of these prohormones and their processing proteases into dense core secretory granules (DCSGs) that the precursors are cleaved to generate their active forms. Prohormone convertase (PC)1/3 is a processing protease that is targeted to DCSGs. The signal for targeting PC1/3 to DCSGs resides in its carboxy-terminal tail (PC1/3617–753), where 3 regions (PC1/3617–625, PC1/3665–682, and PC1/3711–753) are known to aid in sorting and membrane association. In this article, we have determined a high-resolution structure of the extreme carboxy-terminal sorting domain, PC1/3711–753 in micelles by NMR spectroscopy. PC1/3711–753 contains 2 alpha helices located between residues 722–728 and 738–750. Functional assays demonstrate that the second helix (PC1/3738–750) is necessary and sufficient to target a constitutively secreted protein to granules, and that L745 anchors a hydrophobic patch that is critical for sorting. Also, we demonstrate that calcium binding by the second helix of PC1/3711–753 promotes aggregation of the domain via the hydrophobic patch centered on L745. These results provide a structure-function analysis of a DCSG-sorting domain, and reveal the importance of a hydrophobic patch and calcium binding in controlling the sorting of proteins containing alpha helices to DCSGs.

Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.

The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.

Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.