Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA–protein complexes with nucleotide resolution in living cells.     

Mechanism of translation based on intersubunit complementarities of ribosomal RNAs and tRNAs

A universal rule is found about nucleotide sequence complementarities between the regions 2653-2666 in the GTPase-binding site of 23S rRNA and 1064-1077 of 16S rRNA as well as between the region 1103-1107 of 16S rRNA and GUUCG (or GUUCA) of tRNAs. This rule holds for all species in the living kingdoms except for two protista mitochondrial rRNAs of Trypanosoma brucei and Plasmodium falciparum. We found that quite similar relationships for the two species hold under the assumption presented in the present paper. The complementarity between T-loop of tRNA and the region 1103-1107 of 16S rRNA suggests that the first interaction of a ribosome with aminoacyl-tRNAEF-TuGTP ternary complex or EF-GGDP complex could occur at the region 1103-1107 of 16S rRNA with the T-loop-D-loop contact region of the ternary complex or the domain IV-V bridge region of the EF-GGDP complex. The second interaction should occur between the A-site codon and the anticodon loop or between the anticodon stem/loop of A-site tRNA and the tip of domain IV of EF-G. The above stepwise interactions would facilitate the collision of the region 1064-1077 of 16S rRNA with the region around A2660 at the alpha-sarcin/ricin loop of 23S rRNA. In this way, the universal rule is capable of explaining how spectinomycin-binding region of 16S rRNA takes part in translocation, how GTPases such as EF-Tu and EF-G can be introduced into their binding site on the large subunit ribosome in proper orientation efficiently and also how driving forces for tRNA movement are produced in translocation and codon recognition. The analysis of T-loops of all tRNAs also presents an evolutionary trend from a random and seemingly primitive sequence, as defined to be Y type, to the most developed structure, such as either 5G7 or 5A7 types in the present definition.     

Structure of free Thermus flavus 5 S rRNA at 1.3 nm resolution from synchrotron X-ray solution scattering

The shape of free Thermus flavus 5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initio simulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. The atomic models of the 5 S rRNA domains A-D-E and B-C in the form of elongated helices can be well accommodated within the shape, yielding a tentative model of the structure of the free 5 S rRNA in solution. Its comparison with the recent protein-RNA map in the ribosome (Svergun, D. I., and Nierhaus, K. H. (2000) J. Biol. Chem. 275, 14432-14439) indicates that the 5 S rRNA becomes essentially more compact upon complex formation with specific ribosomal proteins. A conceivable conformational change involves rotation of the B-C domain toward the A-D-E domain. The model of free 5 S rRNA displays no interactions between domains E and C, but such interactions are possible in the bound molecule.     

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.     

Application of a nuclease from rye nucleus for structural studies of plant ribonucleic acids.

A new nuclease (Rn) isolated from rye nucleus was applied for the structural studies of methionine initiator transfer ribonucleic acid and ribosomal 5S rRNA from yellow lupin seeds. The enzyme shows high specificity for some regions of both RNAs. The dihydrouridine and ribothymidine loops which are supposed to be involved in the tertiary interactions of the methionine initiator tRNA were hydrolysed. The anticodon loop is not digested at all. 5S rRNA was digested in single stranded regions (loops). The cleavage pattern of the tRNA and 5S rRNA obtained with Rn enzyme, suggests not only the high specificity toward single stranded regions, but also some dependence on their tertiary structure.     

Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA^Asp analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3′-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.     

Detecting the coevolution of biosequences--an example of RNA interaction prediction

A probabilistic graphical model is proposed in order to detect the coevolution between different sites in biological sequences. The model extends the continuous-time Markov process of sequence substitution for single nucleic or amino acids and imposes general constraints regarding simultaneous changes on the substitution rate matrix. Given a multiple sequence alignment for each molecule of interest and a phylogenetic tree, the model can predict potential interactions within or between nucleic acids and proteins. Initial validation of the model is carried out using tRNA and 16S rRNA sequence data. The model accurately identifies the secondary interactions of tRNA as well as several known tertiary interactions. In addition, results on 16S rRNA data indicate this general and simple coevolutionary model outperforms several other parametric and nonparametric methods in predicting secondary interactions. Furthermore, the majority of the putative predictions exhibit either direct contact or proximity of the nucleotide pairs in the 3-dimensional structure of the Thermus thermophilus ribosomal small subunit. The results on RNA data suggest a general model of coevolution might be applied to other types of interactions between protein, DNA, and RNA molecules.     

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation.

Lim and Spirin [25] proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation. Spirin and Lim [26] excluded the possibilities of the side-by-side model proposed by Johnson et al [13] and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus [3]. However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge. With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al [19]. In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA. The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation. The A76 of A site tRNA overlaps the binding region of puromycin. The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA [14] and 17 A from the 5' end of the anticodon of A site tRNA [15]. It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally [6]. A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model.     

Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA

The site of Escherichia coli 16S ribosomal RNA cross-linked to the 5'-anticodon base of A site bound E. coli valyl-tRNA was identified. Cross-linking was via the affinity probe 6-[(2-nitro-4-azidophenyl)amino]caproate (NAK) or 3-[[2-[(2-nitro-4-azidophenyl)amino]ethyl]dithio]propionate (SNAP) attached to the carboxyl group of the 5'-anticodon base 5-(carboxyethoxy)uridine via an ethylenediamine spacer [Gornicki, P., Ciesiolka, J., & Ofengand, J. (1985) Biochemistry (preceding paper in this issue)]. With both probes, RNase T1 digestion of the isolated 16S RNA-tRNA covalent complex, 5'-32P postlabeling, and gel electrophoresis yielded two oligonucleotides larger than any fragments from non-cross-linked tRNA or rRNA. Appearance of the oligomers was dependent on the presence of the probe on the tRNA. Unmodified tRNA in the A and/or P sites did not yield any product. The presence of elongation factor Tu in the incubation mixture was also required. Dithiothreitol (DDT) treatment of the SNAP-induced covalent complex prior to electrophoresis also abolished the oligomers. Only the larger of the two oligomers (present in a 3:1 ratio) was sequenced. The SNAP dimer was cleaved with DTT, and the rRNA and tRNA oligomers were separated and sequenced as monomers. The NAK dimer was sequenced without cleavage by taking advantage of the differences in electrophoretic mobility among sequence and/or composition isomers of the same length. In both cases, the rRNA oligomer was identified as UACACACCG1401, and the nucleotide cross-linked was shown to be the C1400 residue. The expected tRNA modification site was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.     

Association Thermodynamics and Conformational Stability of β-Sheet Amyloid β(17-42) Oligomers: Effects of E22Q (Dutch) Mutation and Charge Neutralization

Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-atom molecular dynamics, molecular mechanics combined with solvation analysis by statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D-RISM-KH) in a new MM-3D-RISM-KH method to study conformational stability, and association thermodynamics of small wild-type Aβ_17-42 oligomers with different protonation states of Glu²², as well the E22Q (Dutch) mutants. The association free energy of small β-sheet oligomers shows near-linear trend with the dimers being thermodynamically more stable relative to the larger constructs. The linear (within statistical uncertainty) dependence of the association free energy on complex size is a consequence of the unilateral stacking of monomers in the β-sheet oligomers. The charge reduction of the wild-type Aβ_17-42 oligomers upon protonation of the solvent-exposed Glu²² at acidic conditions results in lowering the association free energy compared to the wild-type oligomers at neutral pH and the E22Q mutants. The neutralization of the peptides because of the E22Q mutation only marginally affects the association free energy, with the reduction of the direct electrostatic interactions mostly compensated by the unfavorable electrostatic solvation effects. For the wild-type oligomers at acidic conditions such compensation is not complete, and the electrostatic interactions, along with the gas-phase nonpolar energetic and the overall entropic effects, contribute to the lowering of the association free energy. The differences in the association thermodynamics between the wild-type Aβ_17-42 oligomers at neutral pH and the Dutch mutants, on the one hand, and the Aβ_17-42 oligomers with protonated Glu²², on the other, may be explained by destabilization of the inter- and intrapeptide salt bridges between Asp²³ and Lys²⁸. Peculiarities in the conformational stability and the association thermodynamics for the different models of the Aβ_17-42 oligomers are rationalized based on the analysis of the local physical interactions and the microscopic solvation structure.     

Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine

5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N¹-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA^Phe at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent ‘loosening’ of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.     

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.     

Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective.

The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction

Background

A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1.

Results

The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases.

Conclusion

Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.