Studies on transfer ribonucleic acids and related compounds. XXXII. Synthesis of ribonucleotides corresponding to residues 1-5 and 6-10 of tRNAfMet from E. coli and their base conversion analogs.

E. Coli tRNAfMet fragments, C-G-C-G-Gp (bases 1-5), U-G-C-G-Gp (base 1 transition, analog) pG-G-C-G-Gp (base 1 transversion analog) and pG-G-s4U-G-Gp (bases 6-10) were synthesized by triester methods using 2'-O-(o-nitrobenzyl) nucleotides including a 3',5'-bisphosphorylated guanosine derivative. The s4U containing pentanucleotide was derived from the pG-G-C-G-Gp by treatment with liquid hydrogen sulfide.     

Synthesis of the nascent strand of tRNAfMet from E coli.

Oligonucleotides corresponding to the total tRNAfMET FROM E. coli have been synthesized. These fragments were joined by using RNA ligase to yield quarter molecules. The 5'-quarter molecule showed 84% amino acid acceptor activity when it was combined with the natural three quarter molecule.     

Purification of ten transfer ribonucleic acids from E. coli K-12 MO

A flowsheet has been developed which leads to the purification of 10 transfer ribonucleic acids (tRNAs) from E. coli K-12 MO. Crude tRNAs were recovered by phenol extraction and two ethanol precipitation steps. The initial separation of the crude tRNA mixture was achieved by RPC-3 reversed-phase chromatography. Following rechromatography under other conditions, the following tRNAs were recovered at a purity of 70 to 100%: arginine, aspartic acid, glutamic acid, lysine, formylmethionine-1, formylmethionine-3, normal methionine, phcnylalanine-1, phenylalaninc-2, and valine. Recoveries for these tRNAs ranged from 15 to 60%. The flowsheet was demonstrated with chromatogaphic runs ranging from 1,300 to 250,000 A₂₆₀ units per run. The chromatographic steps were simple and readily reproducible.     

Large scale production of transfer ribonucleic acids from E. coli K-12 MO7

An engineering-scale procedure for the recovery of 300–400 g batches of mixed transfer ribonucleic acids is described. Semicontinuous growth of E. coli K-12 MO7 yielded 77 kg of harvested cells in four days. Phenol extraction and ethanol precipitation recovered a crude tRNA material that was further purified by DKAE-cellulose chromatography in runs of 1 × 10⁶ A₂₆₀ units each on a 6 × 30 in. column using a 240 1, gradient elution. The purified tRNAs were partially concentrated and resolved into three groups.     

Studies on transfer ribonucleic acids and related compounds. XLV. Block condensation of ribooligonucleotides containing 2'-O-tetrahydrofuranyl-5'-O-dimethoxytritylnucleosides.

2'-O-Tetrahydrofuranyl-5'-O-dimethoxytrityl-N-protected nucleosides were phosphorylated to give the 3'-(o-chlorophenyl) phosphates which were then condensed with 3',5'-unprotected nucleosides to elongate the chain in the 3'-direction. The 5'-dimethoxytrityl group of these oligonucleotides was selectively deblocked by treatment with zinc bromide. The rate of removal of the dimethoxytrityl group differed in each nucleotide. A dodecamer containing a termination codon UAG, U(AGU)3AG, was synthesized by elongating the chain in the 5'-direction using the selective dedimethoxytritylation followed by condensation of protected oligonucleotide blocks.     

Studies on transfer ribonucleic acids and related compounds. XXVI. Circular dichroic properties of cyclic oligoribonucleotides and their linear counterparts

The CD spectra of cUpUp, cCpCp, and cGpGp derived from DCC-catalyzed polymerization of the relevant protected ribonucleoside 3′-phosphates are described. Similar studies on Up, U > p, and cUp, as well as cUpUpUp and cUpUpUpUp, are presented. The spectral properties of the cyclic oligomers are compared with those of the corresponding linear oligomers with terminal 3′-phosphates so as to demonstrate that disruption of normal right-handed base stacking is considerable in these RNA loops.     

Studies on transfer ribonucleic acids and related compounds. IX. Ribooligonucleotide synthesis using a photosensitive o-nitrobenzyl protection at the 2'-hydroxyl group

o-Nitrobenzyl group was introduced to the 2′-hydroxyl function of uridine via 2′,3′-O-(dibutylstannylene) uridine. The benzylated uridine was protected at the 5′-hydroxyl group with monomethoxytrityl chloride and condensed with 2′,3′-O-dibenzoyluridine 5′-phosphate or N,N′,2′,3′-O-tetrabenzoyladenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). o-Nitrobenzyl ether linkage of the dinucleotides was removed by UV irradiation with wavelength longer than 320 nm. Deprotected UpU and UpA thus obtained were characterized by RNase A digestion.     

Studies on transfer ribonucleic acids and related compounds. VIII(1). Further studies on aromatic phosphoramidates as a protecting group for phosphomonoesters

Stability of aromatic phosphoramidates was studied using 2',3'-O-dibenzoyluridine 5'-phosphoramidates and N,2',3'-O-tribenzoylcytidine 5'-phosphate. The effect of dicyclohexylcarbodiimide in this mixture was investigated. Decomposition of the anilidate was slower in the presence of DCC.Substituted anilidates of uridine 5'-phosphate were synthesized and the stability of these amidates in anhydrous pyridine was studied.2'-O-Benzoyluridine 3'-phosphoranilidate and the corresponding beta-naphthylidate were compared in their stabilities in anhydrous pyridine, 50% aqueous pyridine and 80% acetic acid. 2'-O-Benzoyluridine 3'-phosphoro-beta-naphthylidate was used for synthesis of dinucleotides.     

The nucleotide sequences of the two glutamine transfer ribonucleic acids from Escherichia coli.

The nucleotide sequences of the two glutamine tRNA species in Escherichia coli K12 have been determined. Sufficient data was obtained to order unambiguously the products of complete RNase digestion of tRNA2Gln, and all but one oligonucleotide from tRNA1Gln. The sequence of tRNA1Gln was established by analogy with tRNA1Gln, as the two tRNAs are very similar, differing by only 7 residues out of 75. tRNA1Gln has the anticodon NUG, where N is a modified nucleotide which is likely to be a derivative of 2-thiouridine, and is specific for the codon CAA. tRNA1Gln has the anticodon CUG, and is specific for the codon CAG (Folk, W. R., and Yaniv, M. (1972) Nature 237, 165). The complete sequences of the tRNAGln species are: See journal for formula (Unique residues are enclosed in parentheses, with the residue in tRNA1Gln above that in tRNA2Gln.).