We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

An algorithm is presented which solves the problem of obtaining a rigorous helicoidal description of an irregular nucleic acid segment. Central to this approach is the definition of a function describing simultaneously the curvature of the nucleic acid segment in question and the corresponding stepwise variation of helicoidal parameters along the segment. Minimisation of this function leads to an optimal distribution of the conformational irregularity of the segment between these two components. Further, it is shown that this approach can be applied equally easily to single or double stranded nucleic acids. The results of this analysis yield both the absolute helicoidal parameters of individual bases/base pairs and the relative helicoidal parameters between successive bases/base pairs as well as the overall locus of the helical axis. The possibilities of this mathematical approach are demonstrated with the help of a computer program termed "Curves" which is applied to the study of a number of different nucleic acid structures.

The theory is proposed that the structure of the genetic code was determined by the sequence of evolutionary emergence of new amino acids within the primordial biochemical system.

The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3;(ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is

The anticodon has previously been shown to play a role in recognition of certain transfer RNAs by aminoacyl-tRNA synthetases; however, the extent to which this sequence dictates tRNA identity is generally unknown. To investigate the contribution of the anticodon to the identity of Escherichia coli methionine and valine tRNAs, in vitro transcripts of these tRNAs were prepared that contained normal and interchanged anticodon sequences. Transcripts containing wild-type tRNA sequences were excellent substrates for their respective cognate aminoacyl-tRNA synthetases and were effectively discriminated against by a variety of noncognate enzymes. The mutant tRNAs produced by switching the anticodon sequences lost their original tRNA identity and assumed an identity corresponding to the acquired anticodon sequence. These results indicate that the anticodon contains sufficient information to distinguish methionine and valine tRNAs with high fidelity.

The nucleotide sequence of an RNA primer molecule for initiation of Rous sarcoma virus DNA synthesis in vitro has been determined. The sequence can be drawn in a cloverleaf structure typical of tRNAs with an anticodon for tryptophan. Aminoacylation of the molecule confirms that it is tRNA-Trp. The same sequence and aminoacylation results are obtained regardless of whether the RNA is isolated from virions or from cells of chickens, the natural host for this virus. It is the only species of tRNA-Trp that is dectected in chicked cell tRNA.

One of the eight endonuclease EcoRI fragments of yeast DNA that hybridize to yeast tRNATyr has been identified with the genetically defined nonsense-suppressor locus SUP4. This identification was achieved by analyzing the meiotic linkage between the genetic determinant for the SUP4 phenotype and that for an electrophoretic variant of the EcoRI fragment. The SUP4 gene was then cloned from an ochre-suppressing yeast strain and analyzed by DNA sequencing. A wild-type SUP4 gene and two other genetically unidentified tRNATyr genes were also sequenced. The sequence of the ochre suppressor differs from that of the wild-type genes by virtue of a G.C leads to T.A transversion in the base pair that codes for the wobble position base of the tRNATyr anticodon. All four genes contain, immediately to the 3' side of the anticodon triplet, a 14 base pair tract that is not present in mature tRNATyr. Although the four genes, which represent three unlinked chromosomal loci, all encode the same mature tRNA sequence, there is virtually no observable sequence homology between the three loci in the region preceding the 5' end of the mature tRNATyr sequences.

The structure of yeast transfer RNA aspartic acid has been refined in one crystal form to 3 A resolution using the restrained least-squares method of Hendrickson and Konnert and real-space fitting using the FRODO program of Jones. The final crystallographic discrepancy index R is 23.5% for 4585 reflections with magnitudes twice their standard deviations between 10 and 3 A. With lower occupancies for some residues of the D-loop, the phosphate U1, and the base U33, the R-factor is 22.3%. The adaptation of the restrained least-squares program for nucleic acids and the progress of the refinement are described. The conformations are analysed with respect to stereochemistry and folding of the backbone. The contacts and hydrogen bonds of the secondary structure are compared with those of yeast tRNAPhe. The presence of only four bases in the variable loop, instead of five as in yeast tRNAPhe, leads to a rotation of residue 48 and a lateral movement of residue 46. These two rearrangements induce different environments for [U8 ... A14]... A21 as well as for A9 and G45. Otherwise, all tertiary contacts observed in yeast tRNAPhe are present in yeast tRNAAsp, except for the absence of hydrogen-bonding between G18 of the D-loop and C56 of the T-loop. The presence of anticodon triplet pairing leads to a distribution of temperature factors different from that observed in yeast tRNAPhe with a stabilization of the AC stem-and-loop and a destabilization of the T and D-loops. We are inclined to suggest that the labilization of the interactions between the T and D-loops is a consequence of the interaction of the anticodon triplets of symmetry-related molecules through hydrogen bonding, which mimics the interaction between the anticodon and its cognate codon on the messenger RNA.