In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3′ terminus of the intron (ωG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/ωG) including the GBS and ωG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, ωG is recognized by the formation of a base triple with the G264[bull ]C311 base pair, and this recognition is stabilized by the stacking interaction between ωG and C262. The bulged structure at A263 causes a large helical twist angle (40 ± 8°) between the G264[bull ]C311 and C262[bull ]G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a “Bulge-and-Twist” (BT) pocket. With another twist angle between the C262[bull ]G312 and G413[bull ]C313 base pairs (45 ± 10°), the axis of GBS/ωG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 Å resolution electron density map (Golden et al., Science, 1998, 282:345–358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

Hydroxyl radical footprinting is a powerful technique often employed in characterization of the tertiary interactions between proteins and nucleic acids. Following the generation of a nucleic acid “ladder” either by chemical or enzymatic reactions, the radiolabeled products are traditionally separated by denaturing gel electrophoresis and further quantified by phosphorimaging techniques. Here we report the use of ion pair reverse phase liquid chromatography to analyze the products of an RNA footprinting reaction using fluorescently labeled RNA molecules. This technique offers several advantages over existing procedures, including rapid analysis, automation, and direct quantification of the cleavage products without the need to employ radiolabeling. To illustrate the resolving power of this technique, we have analyzed the products of base hydrolysis, generated from a fluorescently labeled RNA molecule and have subsequently used this method to define the solvent accessibility of the substrate strand as it docks with the hairpin ribozyme.

Prp2p, Prp16p, Prp22p, and Prp43p are members of the DEAH-box family of ATP-dependent putative RNA helicases required for pre-mRNA splicing in Saccharomyces cerevisiae. Recently, mammalian homologues of Prp43p and Prp22p have been described, supporting the idea that splicing in yeast and man is phylogenetically conserved. In this study, we show that a murine cell line resistant to the novel immunoregulatory drug Leflunomide (AravaTM) overexpresses a 135-kDa protein that is a putative DEAH-box RNA helicase. We have cloned the human counterpart of this protein and show that it shares pronounced sequence homology with Prp16p. Apart from its N-terminal domain, which is rich in RS, RD, and RE dipeptides, this human homologue of Prp16p (designated hPrp16p) is 41% identical to Prp16p. Significantly, homology is not only observed within the phylogenetically conserved helicase domain, but also in Prp16p-specific sequences. Immunofluorescence microscopy studies demonstrated that hPrp16p co-localizes with snRNPs in subnuclear structures referred to as speckles. Antibodies specific for hPrp16p inhibited pre-mRNA splicing in vitro prior to the second step. Thus, like its yeast counterpart, hPrp16p also appears to be required for the second catalytic step of splicing. Taken together, our data indicate that the human 135-kDa protein identified here is the structural and functional homologue of the yeast putative RNA helicase, Prp16p.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5′ exon before the first step of splicing and with the 5′ and 3′ exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5′ and 3′ exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5′ exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5′ exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5′ splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5′ exon in two steps during its targeting to the 5′ splice site.

Self-splicing of the Tetrahymena pre-rRNA is inhibited by a conserved rRNA hairpin P(−1) upstream of the 5′ splice site. P(−1) inhibits self-splicing by competing with formation of the P1 splice site helix. Here we show that the P(−1) hairpin also enhances dissociation of the spliced products, which was monitored by native gel electrophoresis. Mutations that stabilize the rRNA hairpin increase the rate of dissociation approximately 10-fold, from 0.5 min−1 for the wild-type RNA to ∼4 min−1 at 30 °C. Conversely, mutations or oligonucleotides that inhibit refolding of the exons and that stabilize the P1 helix decrease the rate of product release. The results suggest that refolding of products can be used to stimulate the turnover of ribozyme-catalyzed reactions. In the pre-rRNA, this conformational change helps shift the equilibrium of self-splicing toward the mature rRNA.

Specific aminoacylation by aminoacyl-tRNA synthetases requires accurate recognition of cognate tRNA substrates. In the case of alanyl-tRNA synthetase (AlaRS), RNA duplexes that mimic the acceptor stem of the tRNA are efficient substrates for aminoacylation in vitro. It was previously shown that recognition by AlaRS is severely affected by a simple base pair transversion of the G2:C71 pair at the second position in the RNA helix. In this study, we determined the aminoacylation efficiencies of 50 variants of the tRNAAla acceptor stem containing substitutions at the 2:71 position. We find that there is not a single functional group of the wild-type G2:C71 base pair that is critical for positive recognition. Rather, we observed that base-pair orientation plays an important role in recognition. In particular, pyrimidine2:purine71 combinations generally resulted in decreased aminoacylation efficiency compared to the corresponding purine:pyrimidine pair. Moreover, the activity of a pyrimidine:purine variant could be partially restored by the presence of a major groove amino group at position 71. In an attempt to understand this result further, dielectric continuum electrostatic calculations were carried out, in some cases with additional inclusion of van der Waals interaction energies, to determine interaction potentials of the wild-type duplexAla and seven 2:71 variants. This analysis revealed a positive correlation between major groove negative electrostatic potential in the vicinity of the 3:70 base pair and measured aminoacylation efficiency.

In recent years, a growing number of mammalian genes have been shown to undergo alternative splicing in response to extracellular stimuli. However, the factors and pathways involved in such signal-induced alternative splicing are almost entirely unknown. Here we describe a novel method for identifying candidate trans-acting factors that are involved in regulating mammalian alternative splicing, using the activation-induced alternative splicing of the human CD45 gene in T cells as a model system. We generated a cell line that stably expresses a CD45 minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the endogenous CD45 gene. Following mutagenesis of this cell line, and multiple rounds of selection for cells that displayed aberrant levels of GFP expression, we isolated several cell lines that are at least partially defective in their ability to support regulated alternative splicing of endogenous CD45 pre-mRNA in response to cell stimulation. Thus we have successfully isolated mutants in a mammalian alternative splicing pathway through use of a somatic cell-based genetic screen. This study clearly demonstrates the feasibility of using genetic screens to further our understanding of the regulation of mammalian splicing, particularly as it occurs in response to environmental cues.

The recent rapid advances that have been made both in cryo-electron microscopy (cryo-EM) and X-ray crystallography of bacterial ribosomes or their subunits (e.g., Stark et al., 1997a, 1997b; Ban et al., 1998; Malhotra et al., 1998) have led to a correspondingly rapid advance in our understanding of the three-dimensional (3D) arrangement in situ of the ribosomal RNA and protein molecules. In 1997 we published a model for the 16S rRNA (Mueller & Brimacombe, 1997a), which was fitted to a cryo-EM reconstruction at 20 Å resolution of the Escherichia coli 70S ribosome carrying tRNAs at the ribosomal A and P sites (Stark et al., 1997a). Subsequently, on the basis of the available RNA–protein interaction data (Mueller & Brimacombe, 1997b), we were able to fit the structure of ribosomal protein S7 as determined by X-ray crystallography (Hosaka et al., 1997; Wimberly et al., 1997) into our model in such a way as to satisfy both the biochemical data and the electron density of the EM reconstruction (Tanaka et al., 1998). More recently, the 16S model has been refined to fit an EM reconstruction at 13 Å resolution (Brimacombe et al., 2000), the latter being itself a refinement of the published EM reconstruction at 18 Å (Stark et al., 1997b) of 70S ribosomes carrying an EF-Tu/tRNA ternary complex stalled with the antibiotic kirromycin. In the refined 16S model, only minor changes needed to be made in the arrangement of the rRNA region interacting with S7 and in the positioning of the protein itself.

The crystal structure of the 19-mer RNA, 5′-GAAUGCCUGCGAGCAUCCC-3′ has been determined from X-ray diffraction data to 1.6 Å resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A[bull ]C mismatch and one A[bull ]C/G[bull ]U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson–Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A[bull ]C mismatch may reveal a unique structural motif required for the function of Com.

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 5′ end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5′ end of deacylated tRNAPhe to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 45° between the respective planes of the molecules. The terminal phosphates of 3′CCA are separated by 23 Å when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 Å side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 Å distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 Å from the 4′ position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

We present a novel missense suppression system for the selection of tRNA2Gln mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA2Gln mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C → U at position 34 and U → C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA2Gln in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.

Primary transcripts made by RNA polymerase II (Pol II), but not Pol I or Pol III, are modified by addition of a 7-methylguanosine (m7G) residue to the triphosphate 5′ end shortly after it emerges from the polymerase. The m7G “caps” of small nuclear and small nucleolar RNAs, but not messenger RNAs, are subsequently hypermethylated to a 2,2,7-trimethylguanosine (TMG) residue. U6 RNA, the only small nuclear RNA synthesized by Pol III in most eukaryotes, does not receive a methylguanosine cap. However, human U6 RNA is O-methylated on the 5′-terminal (γ) phosphate by an enzyme that recognizes the 5′ stem-loop of U6. Here we show that variant yeast U6 RNAs truncated or substituted within the 5′ stem-loop are TMG capped in vivo. Accumulation of the most efficiently TMG-capped U6 RNA variant is strongly inhibited by a conditional mutation in the largest subunit of Pol III, confirming that it is indeed synthesized by Pol III. Thus, methylguanosine capping and cap hypermethylation are not exclusive to Pol II transcripts in yeast. We propose that TMG capping of variant U6 RNAs occurs posttranscriptionally due to exposure of the 5′ triphosphate by disruption of protein binding and/or γ-methyl phosphate capping. 5′ truncation and TMG capping of U6 RNA does not appear to affect its normal function in splicing, suggesting that assembly and action of the spliceosome is not very sensitive to the 5′ end structure of U6 RNA.

NanGIR1 is a member of a new class of group I ribozymes whose putative biological function is site-specific hydrolysis at an internal processing site (IPS). We have previously shown that NanGIR1 requires 1 M KCl for maximal activity, which is nevertheless slow (0.03 min–1). We used in vitro selection and an RNA pool with approximately nine mutations per molecule to select for faster hydrolysis at the IPS in 100 mM KCl. After eight rounds of selection, GIR1 variants were isolated that catalyzed hydrolysis at 300-fold greater rates than NanGIR1 RNA. Although not required by the selection, many of the resultant RNAs had increased thermal stability relative to the parent RNA, and had a more compact structure as evidenced by their faster migration in native gels. Although a wide spectrum of mutations was found in generation 8 clones, only two mutations, U149C and U153C, were common to greater than 95% of the molecules. These and one other mutation, G32A, are sufficient to increase activity 50-fold. All three mutations lie within or proximal to the P15 pseudoknot, a structural signature of GIR1 RNAs that was previously shown to be important for catalytic activity. Overall, our findings show that variants of the Naegleria GIR1 ribozyme with dramatically improved activity lie very close to the natural GIR1 in sequence space. Furthermore, the selection for higher activity appeared to select for increased structural stability.

Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl–tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNAArg can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNAArg isoacceptors and heterologous tRNAAsp, we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination.

Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides. Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions. Here we discuss conserved RNA folding pathways that are revealed by covariation patterns. In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners. Such “coupled” covariations between three or more nucleotides were found in various types of RNAs. The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases. Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts.

Escherichia coli ribosomal protein L4 autogenously regulates transcription of the S10 operon, which encodes L4 and 10 other ribosomal proteins. Regulation results from L4-stimulated premature transcription termination at a U-rich site in the untranslated leader. The process requires transcription factor NusA. Here we report a detailed analysis of the RNA requirements for NusA-dependent, L4-mediated transcription control. We found that efficient regulation requires multiple features of the S10 leader, including two hairpins, called HD and upper HE, a connecting tether, and a U-rich sequence at the distal side of HE. As expected, regulation was optimal when all 7 Us were maintained in the U4CGU3 sequence at the termination site. However, despite the apparent specificity of L4 action on only the S10 operon, there is surprising flexibility at the primary sequence level for the HD-tether-HE region. Changes in the sequence of non-base-paired nucleotides flanking the HD hairpin or an A at the second position of the HD loop reduced L4 regulation, but other changes had little or no effect. Furthermore, generic hairpins from other RNAs could replace the natural HD and upper HE hairpins with little or no reduction of L4 control, suggesting that the secondary structure elements are also relatively generic. The lack of specific sequence requirements suggests that L4 may recognize multiple elements within this region of the nascent leader.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3′ end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3′ end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

In yeast, the 3′ end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position −5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position −2), whereas sequence changes at position −1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3′ end of 18S rRNA and the 5′ end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.