Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado47-containing tRNAP41t species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoramidites. p-Nitrophenylethyl groups were used to protect the o' of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA e't containing 7-methylguanosine at position 47.Ribonucleic acids (RNAs) play a wide range of important roles in living cells. These include the traditional roles in transcription and translation (mRNA, tRNA, and rRNA) of genetic information. Excitement in RNA chemistry has grown with the discovery of RNA sequences with catalytic activity (1), the formation of "lariats" during the processing of pre-RNA (2), and the development of recombinant RNA technology (3, 4). RNA sequences of the tRNA type are required in the first step of chlorophyll biosynthesis (5). tRNAs are particularly attractive as synthetic targets because their length, 70-85 nucleotides, is sufficient to demonstrate a successful chemical procedure. In addition, they have specific biological functions that can be demonstrated in the synthetic product. They are also interesting molecules in their own right, having been implicated in gene expression (6), in propagation of tumor viruses (7), and in cellular differentiation and development in cancer induction (8-10). The chemical synthesis of RNA molecules allows complete flexibility in the choice of sequence and the ability to introduce modifications at specific sites. Thus, the ability to chemically synthesize molecules of this size should permit a rapid increase in the rate of understanding of the full biological roles of RNA molecules paralleling that which occurred earlier when the chemical synthesis of DNA oligomers became routine.Our laboratory has had as a major objective the development of methods for the chemical synthesis ofRNA sequence (11,12). These methods have now progressed to the point where the synthesis of 70-to 80-unit molecules is a practical possibility. To demonstrate the chemical method, a sequence was chosen that was identical to that of an Escherichia coli Ado-47 tRNAP et species (i.e., containing adenosine instead of 7-methylguanosine at position 47), except th...

We have determined the nucleotide sequence of the major species of E. coli tRNASer and of a minor species having the same GGA anticodon. These two tRNAs should recognize the UCC and UCU codons, the most widely used codons for serine in the highly expressed genes of E. coli. The two sequences differ in only one position of the D-loop. Neither tRNA has a modified adenosine in the position 3'-adjacent to the anticodon. This can be rationalized on the basis of a structural constraint in the anticodon stem and may be related to optimization of the codon-anticodon interaction. Comparison of all E.coli serine tRNAs (and that encoded by bacteriophage T4) reveals characteristic (possibly functional) features. Evolutionary analysis suggests an eubacterial origin of the T4 tRNASer gene and the existence of a recent common ancestor for the tRNASerGGA and tRNASerGUC genes.

Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNAMet. Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary structural rules. Comparison of cleavage patterns of yeast initiator tRNAM", tDNAMc' (a DNA oligomer having the sequence of tRNA'"') and the anti-tDNAM"' (the complement of tDNAM"') suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhuI and CfoI restriction endonucleases at two GCGjC sites which would be in double-stranded rcgions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.

The effect of U(34) dethiolation on the anticodon-anticodon association between E. coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique. An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C. The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5. Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.

The sequence of three tRNAs from Halobacterium cutirubrum have been determined. The sequences of tRNAValGAC and tRNAValCAC differ by only one nucleotide which is in the 5' terminal anticodon position. These tRNAs as well as that of tRNAAlaCGC are compared to other known halobacterial tRNAs. An observed paucity (or absence) of U in the first anticodon position is unique to archaebacterial tRNAs and may be indicative of unusual decoding properties of these organisms.