The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited.

The crystal structure of the monoclinic form of yeast phenylalanine tRNA has been redetermined at a resolution of 1.93 A. The structure of yeast tRNAphe described here is more accurate than its predecessors not only because it incorporates higher resolution data, but also because it has been refined using techniques that had not been developed when its predecessors were determined more than 20 years ago. The 1.93 A resolution version of this structure differs interestingly from its predecessors in its details. In loop regions particularly, the backbone torsion angles in the new structure are not the same as those reported earlier. Several new divalent cation binding sites have been identified, and the water structure that has emerged is also different.

Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation.

Elongation factor (EF) G promotes tRNA translocation on the ribosome. We present three-dimensional reconstructions, obtained by cryo-electron microscopy, of EF-G-ribosome complexes before and after translocation. In the pretranslocation state, domain 1 of EF-G interacts with the L7/12 stalk on the 50S subunit, while domain 4 contacts the shoulder of the 30S subunit in the region where protein S4 is located. During translocation, EF-G experiences an extensive reorientation, such that, after translocation, domain 4 reaches into the decoding center. The factor assumes different conformations before and after translocation. The structure of the ribosome is changed substantially in the pretranslocation state, in particular at the head-to-body junction in the 30S subunit, suggesting a possible mechanism of translocation.

Higher-order structure of bovine mitochondrial tRNA(Phe) lacking the 'conserved' GG and T psi CG sequences as inferred by enzymatic and chemical probing.

Bovine mitochondrial (mt) phenylalanine tRNA (tRNA(Phe)), which lacks the 'conserved' GG and T psi YCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and its higher-order structure was investigated using RNaseT2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNaseT2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected from the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNA(Phe) genes from various organisms, it was inferred that bovine mt tRNA(Phe) lacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNA(Phe) to preserve its L-shaped higher-order structure.

Dynamics of transfer RNAs analyzed by normal mode calculation.

Normal mode calculation is applied to tRNAPhe and tRNAAsp, and their structural and vibrational aspects are analyzed. Dihedral angles along the phosphate-ribose backbone (alpha, beta, gamma, epsilon, zeta) and dihedral angles of glycosyl bonds (chi) are selected as movable parameters. The calculated displacement of each atom agrees with experimental data. In modes with frequencies higher than 130 cm-1, the motions are localized around each stem and the elbow region of the L-shape. On the other hand, collective motions such as bending or twisting of arms are seen in modes with lower frequencies. Hinge axes and bend angles are calculated without prior knowledge. Movements in modes with very low frequencies are combinations of hinge bending motions with various hinge axes and bend angles. The thermal fluctuations of dihedral angles well reflect the structural characters of transfer RNAs. There are some dihedral angles of nucleotides located around the elbow region of L-shape, which fluctuate about five to six times more than the average value. Nucleotides in the position seem to be influential in the dynamics of the entire structure. The normal mode calculation seems to provide much information for the study of conformational changes of transfer RNAs induced by aminoacyl-tRNA synthetase or codon during molecular recognition.

Reproducing the three-dimensional structure of a tRNA molecule from structural constraints.

The three-dimensional structure of yeast tRNA(Phe) was reproduced at atomic resolution with the automated RNA modeling program MC-SYM, which is based on a constraint-satisfaction algorithm. Structural constraints used in the modeling were derived from the secondary structure, four tertiary base pairs, and other information available prior to the determination of the x-ray crystal structure of the tRNA. The program generated 26 solutions (models), all of which had the familiar "L" form of tRNA and root-mean-square deviations from the crystal structure in the range of 3.1-3.8 A. The interaction between uridine-8 and adenosine-14 was crucial in the modeling procedure, since only this among the tertiary pairs is necessary and sufficient to reproduce the L form of tRNA. Other tertiary interactions were critical in reducing the number of solutions proposed by the program.

Cleavage of yeast tRNA(phe) with Ni(III) and Co(III) complexes of bleomycin.

The BLM-Ni(III) complex preferentially cleaves guanine residues at D loop and anticodon loop of yeast tRNA(phe) by aniline treatment, whereas the BLM-Co(III) complex degrades certain 5'-AN sites of the minor groove region after irradiation of UV light. These cleavage features are clearly different from that of the BLM-Fe(II) complex.

Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities.

The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes.

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNAPhe/(U)9/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319-322).

Characterization and preliminary crystallographic studies on large ribosomal subunits from Thermus thermophilus.

Diffracting crystals, suitable for X-ray crystallographic analysis, have been obtained from large (50 S) ribosomal subunits from Thermus thermophilus. These crystals, with P4(1)2(1)2 symmetry and a unit cell of 495 A x 495 A x 196 A, reach typically a size of 0.15 mm x 0.25 mm x 0.35 mm. Using synchrotron radiation at cryo-temperature, these crystals diffract X-rays to better than 9 A resolution, and do not show any measurable decay after a few days of irradiation. They complete a series of crystals, grown by us, from ribosomal particles of the same source, including a 30 S subunits, 70 S ribosomes and complexes of the latter with: (1) an oligomer of 35 uridine residues and (2) the same oligonucleotide together with approximately two Phe-tRNA(Phe) molecules. Crystallographic analysis of the various members of this series should provide information for investigating the conformational changes that take place upon the association of ribosomes from their subunits as well as upon binding of non-ribosomal components that participate in protein biosynthesis.

Crystals of complexes mimicking protein biosynthesis are suitable for crystallographic studies.

A complex of 70S ribosomes from Thermus thermophilus together with an average of 1.5-1.8 equivalents of PhetRNA(Phe) and a short mRNA chain, composed of 35 +/- 5 uridines, was crystallized under the conditions used for the growth of crystals of isolated ribosomes from the same source. Considering the reproducibility of their growth, their internal order and their shape, the crystals of the complex are superior to those of isolated ribosomes. In accord with previous three-dimensional reconstruction and modeling experiments, we conclude that the complex is less flexible and that an average population of complexes is more homogeneous than that of isolated 70S ribosomes. The crystals of the complex diffract to higher than 15 A resolution and can be irradiated with synchrotron X-ray beam at cryo-temperatures for days without noticeable decay. Since the crystals of the complex are apparently isomorphous with these of the isolated 70S ribosomes (P4(1)2(1)2; a = b = 526; c = 315 A), they should provide tool for phasing as well as for locating the mRNA and tRNA binding sites.

Prediction of the three-dimensional structure of Escherichia coli 30S ribosomal subunit: a molecular mechanics approach.

We introduce a computer-assisted procedure for folding large RNA chains into three-dimensional conformations consistent with their secondary structure and other known experimental constraints. The RNA chain is modeled using pseudoatoms at different levels of detail--from a single pseudoatom per helix to a single pseudoatom for each nucleotide. A stepwise procedure is used, starting with a simple representation of the macromolecule that is refined and then extrapolated into higher resolution for further refinement. The procedure is capable of folding different random-walk chains by using energy minimization, allowing generation of a range of conformations consistent with given experimental data. We use this procedure to generate several possible conformations of the 16S RNA in the 30S ribosomal subunit of Escherichia coli by using secondary structure and the neutron-scattering map of the 21 proteins in the small subunit. The RNA chain is modeled using a single pseudoatom per helix. RNA-RNA and RNA-protein crosslinks, reported in current literature, are included in our model. Footprinting data for different ribosomal proteins in the 16S RNA are also used. Several conformations of the 16S RNA are generated and compared to predict gross structural features of the 30S subunit as well as to identify regions of the 16S RNA that cannot be well-defined with current experimental data.

Structure of an unmodified tRNA molecule.

We have used NMR to study the structure of the yeast tRNA(Phe) sequence which was synthesized by using T7 RNA polymerase. Many resonances in the imino 1H- spectrum of the transcript have been assigned, including those of several tertiary interactions. When the Mg2+ concentration is high, the transcript appears to fold normally, and the spectral features of the transcript resemble those of tRNA(Phe). The transcript has been shown to be aminoacylated with kinetics similar to the modified tRNA(Phe) [Sampson, J. R., & Uhlenbeck, O. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1033-1037], suggesting that the structure of the two molecules must be similar. In the absence of Mg2+ or at [tRNA]:[Mg2+] ratios less than 0.2, the transcript does not adopt the native structure, as shown by both chemical shifts and NOE patterns. In these low Mg2+ conditions, a second GU base pair is found, suggesting a structural rearrangement of the transcript. NMR data indicate that the structure of a mutant having G20 changed to U20 is nearly identical with that of the normal sequence, suggesting that the low aminoacylation activity of this variant is not due to a substantially different conformation.

Use of lead(II) to probe the structure of large RNA's. Conformation of the 3' terminal domain of E. coli 16S rRNA and its involvement in building the tRNA binding sites.

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3' domain of the E. coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA. The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections. A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Prediction of three-dimensional structure of Escherichia coli ribosomal RNA.

A model for the tertiary structure of 23S, 16S and 5S ribosomal RNA molecules interacting with three tRNA molecules is presented using the secondary structure models common to E. coli, Z. mays chloroplast, and mammalian mitochondria. This ribosomal RNA model is represented by phosphorus atoms which are separated by 5.9 A in the standard A-form double helix conformation. The accumulated proximity data summarized in Table 1 were used to deduce the most reasonable assembly of helices separated from each other by at least 6.2 A. Straight-line approximation for single strands was adopted to describe the maximum allowed distance between helices. The model of a ribosome binding three tRNA molecules by Nierhaus (1984), the stereochemical model of codon-anticodon interaction by Sundaralingam et al. (1975) and the ribosomal transpeptidation model, forming an alpha-helical nascent polypeptide, by Lim & Spirin (1986), were incorporated in this model. The distribution of chemically modified nucleotides, cross-linked sites, invariant and missing regions in mammalian mitochondrial rRNAs are indicated on the model.

On loop folding in nucleic acid hairpin-type structures.

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations. This model is based on the notion that to a major extent base stacking interactions determine the stability of nucleic acid conformations. The model predicts that loop folding in RNA is characterized by an extension of the base stacking at the 5'-side of the double helix by five or six bases; the remaining gap can then easily be closed by two nucleotides. Conversely, loop folding in DNA is characterized by extending base stacking at the 3'-side of the double helical stem by two or three residues; again bridging of the remaining gap can then be achieved by one or two nucleotides. As an example of loop folding in RNA the anticodon loop of yeast tRNAPhe is discussed. For the DNA hairpin formed by d(ATCCTAT4TAGGAT) it is shown that the loop structure obtained from molecular mechanics calculations obeys the above worded loop folding principles.