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We describe a statistical method to determine if a pair of columns in a multiple alignment of a homologous family of RNA sequences shows evidence of being base paired. The method makes explicit use of a given phylogenetic tree for the sequences in the alignment. It is tested on a multiple alignment of 16S rRNA sequences with good results.
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The core subunit arrangement of alpha 2-beta-beta' within DNA-dependent RNA polymerase holoenzyme alpha 2 beta beta' sigma from Escherichia coli was investigated by neutron small-angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C-bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits, alpha 2, beta and beta' in situ and for the determination of the intersubunit centre-to-centre distances. The contrast between the buffer and the remaining 'hydrogenated' enzyme vanishes if the buffer contains 42% 2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved. The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering intensity differences of the pair-labelled (e.g. both beta and beta' labelled) and both mono-labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains sigma as a dissociable factor, is presented in a three-dimensional model.
In vivo experiments have demonstrated that the ribosomal protein L32 of Saccharomyces cerevisiae brings about the inhibition of splicing of the transcript of its own gene through an RNA structure comprised largely of the first exon. We now show that L32, itself, binds specifically to this RNA. Splicing of the RPL32 transcript in vitro is blocked by the presence of L32. Furthermore, addition of the 75-nucleotide RNA representing the 5' end of the RPL32 transcript stimulates specifically the splicing of the RPL32 substrate, presumably by competing for L32 present in the extract. Use of RNAs carrying mutations shown to abolish the regulation of splicing, either as substrates or as competitors, confirmed that the in vitro reaction is a faithful representation of the situation in vivo. We conclude that the regulation of splicing occurs through the specific binding of L32 to an RNA structure within the first 75 nucleotides of the RPL32 transcript. The RPL32 substrate, bound to L32, forms a complex with U1 snRNP, the first step in spliceosome assembly. The presence of L32 prevents the ATP-dependent association of the U2 snRNP necessary to form a complete spliceosome.
DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.
The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.
The Escherichia coli regulatory protein StpA bears striking similarity to the chromatin-associated protein H-NS. These two proteins have many structural, functional and mechanistic parallels. Although H-NS is more abundant in the cell, both proteins act as transcriptional regulators, both bind to curved DNA and both restrain DNA supercoils. However, StpA is better able to promote RNA annealing and trans-splicing in vitro. In this study, phylogenetic analyses and experiments to examine the protease sensitivity of StpA and H-NS suggest a similar structure for the two proteins. Both proteins consist of two structured domains separated by an exposed protease-sensitive linker. The N-terminal (StpA-NterL) and C-terminal (StpA-CterL) domains of StpA, as well as the full-length StpA and H-NS proteins, were cloned, overproduced in E. coli and purified to homogeneity. StpA-CterL, but not StpA-NterL, promotes strand annealing of complementary RNA oligonucleotides and in vitro trans-splicing of a model group I intron. Both StpA and StpA-CterL exhibited stronger RNA-modulating activity than H-NS. Phylogenetic analyses showed that the N-terminal and C-terminal domains can exist autonomously. The phylogenetic and experimental data are compatible with a two-domain model for StpA and H-NS, with independently functioning modules joined by a non-conserved linker, and with the observed RNA-related activities residing entirely within the C-terminal domain.
L-alpha-amino acids with a nucleobase in the side chain (nucleobase amino acids; NBAs) were used to enhance the function of RNA-binding proteins that recognize structured RNA. These NBAs were utilized in the three-dimensional structure of the protein to enhance RNA binding affinity and specificity as a result of selective recognition of NBAs by RNA bases. NBA units were incorporated at various positions into the HIV-1 nucleocapsid protein NCp7 (residues 1-55), which contains two CCHC-type (Cys-X(2)-Cys-X(4)-His-X(4)-Cys-type; X=an amino acid residue) zinc knuckle domains. The binding ability was evaluated by using the stem-loop (SL)3 region of HIV-1 Psi-RNA. Visible light absorption measurements revealed that two zinc ions bound strongly and quantitatively to the NBA-NCp7 molecule and to the wild-type NCp7 protein. This result indicates that the incorporation of NBA units composed of L-alpha-amino acids did not influence the formation of the specific structure of NCp7. Binding analysis with fluorescein-labeled SL3 RNA revealed that incorporation of NBA units into the NCp7 protein at appropriate positions increased its RNA binding affinity and specificity. An NBA-NCp7 protein that possessed cytosine and guanine NBA units at positions 13 and 46, respectively, showed a binding affinity for SL3 RNA ninefold higher than that of wild-type NCp7 as a result of the specific and cooperative interaction of the NBA units with RNA bases. These results clearly demonstrate that inclusion of NBA units in the three-dimensional structure of an RNA-binding protein is a useful strategy for enhancing the function of the protein.
Interactions between ribosomal protein L11 and a domain of large subunit rRNA have been highly conserved and are essential for efficient protein synthesis. To study the effects of L11 on rRNA folding, a homolog of the Escherichia coli L11 gene has been amplified from Bacillus stearothermophilus DNA and cloned into a phage T7 polymerase-based expression system. The expressed protein is 93% homologous to the L11 homolog from Bacillus subtilis, denatures at temperatures above 72 degrees C, and has nearly identical rRNA binding properties as the Escherichia coli L11 in terms of RNA affinity constants and their dependences on temperature, Mg2+ concentration, monovalent cation, and RNA mutations. Mg2+ and NH4+ are specifically bound by the RNA-protein complex, with apparent ion-RNA affinities of 1.6 mM-1 and 19 M-1, respectively, at 0 degree C. The effect of the thermostable L11 on the unfolding of a 60 nucleotide rRNA fragment containing its binding domain has been examined in melting experiments. The lowest temperature RNA transition, which is attributed to tertiary structure unfolding, is stabilized by approximately 25 degrees C, and the interaction has an intrinsic enthalpy of approximately 13 kcal/mol. The thermal stability of the protein-RNA complex is enhanced by increasing Mg2+ concentration and by NH4+ relative to Na+. Thus L11, NH4+, and Mg2+ all bind and stabilize the same rRNA tertiary interactions, which are conserved and presumably important for ribosome function.
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An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).
In vitro evolution techniques allow RNA molecules with unique functions to be developed. However, these techniques do not necessarily identify the simplest RNA structures for performing their functions. Determining the simplest RNA that binds to a particular ligand is currently limited to experimental protocols. Here, we introduce a molecular-mechanics based algorithm employing molecular dynamics simulations and free-energy methods to predict the minimum sequence requirements for selective ligand binding to RNA. The algorithm involves iteratively deleting nucleotides from an experimentally determined structure of an RNA-ligand complex, performing energy minimizations and molecular dynamics on each truncated structure, and assessing which truncations do not prohibit RNA binding to the ligand. The algorithm allows prediction of the effects of sequence modifications on RNA structural stability and ligand-binding energy. We have implemented the algorithm in the AMBER suite of programs, but it could be implemented in any molecular mechanics force field parameterized for nucleic acids. Test cases are presented to show the utility and accuracy of the methodology.
BACKGROUND: RNA splicing is both ubiquitous and essential for the maturation of precursor mRNA molecules in eukaryotes. The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the 22-nucleotide SL from the SL1 RNA. Very little is known about the various roles that RNA structures play in the complex conformational rearrangements and reactions involved in premRNA splicing. RESULTS: We have determined the solution structure of a domain of the first stem loop of the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMR techniques; this domain contains the splice-donor site and a nine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts a stem-loop structure: nucleotides in the stem region form a classical A-type helix while nucleotides in the hairpin loop specify a novel conformation that includes a helix, that extends for the first three residues; a syn guanosine nucleotide at the turn region; and an extrahelical adenine that defines a pocket with nucleotides at the base of the loop. CONCLUSION: The proximity of this pocket to the splice donor site, combined with the observation that the nucleotides in this motif are conserved among all nematode SL RNAs, suggests that this pocket may provide a recognition site for a protein or RNA molecule in the trans-splicing process.
The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross-linking/label transfer experiments, we have analyzed sequences of the 3'-nontranslated region (3'NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic- and antigenomic-sense transcripts. The results show that the 3'-NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3Dpol-coding sequence and the 3'-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis.
U6 RNA is a key component of the catalytic core of the spliceosome. A metal ion essential for the first catalytic step of pre-mRNA splicing binds to the U80 Sp phosphate oxygen within the yeast U6 intramolecular stem-loop (ISL). Here we present the first structural data for U6 RNA, revealing the three-dimensional structure of the highly conserved U6 ISL. The ISL binds metal ion at the U80 site with the same stereo specificity as the intact spliceosome. The metal-binding site is adjacent to a readily protonated C.A wobble pair. Protonation of the C.A pair and metal binding are mutually antagonistic. These results support a ribozyme model for U6 RNA function and suggest a possible mechanism for the regulation of RNA splicing.
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