RNA CODEWORDS AND PROTEIN SYNTHESIS, II. NUCLEOTIDE SEQUENCE OF A VALINE RNA CODEWORD (1964)

Recent studies demonstrate that synthetic polynucleotides, such as poly U, in- duce with specificity the binding of C14-amino-acyl-sRNA to ribosomes.'-4Char- acteristics of binding in the absence of poly U also have been reported.5C14-Phe-sRNA binding induced by poly U is thought to represent an early step in pro- tein synthesis before peptide bond formation; however, the precise nature of the interaction has not been clarified.To establish the sequence of nucleotides in RNA codewords, we devised a rapid, sensitive method for measuring C'4-amino-acyl-sRNA binding to ribosomes, and have investigated both characteristics of binding and the minimum oligonucleotide chain length required to direct such binding.6pUpUpU, pApApA, and pCpCpC directed the binding of Phe-, Lys-, and Pro-sRNA, respectively, whereas dinucleotides had no effect.Trinucleotides with 5'-terminal phosphate were more active than those with no terminal phosphate, whereas trinucleotides with 2'-(3')-terminal phosphate were inactive.6In this report, trinucleotides of known sequence were used to direct sRNA bind- ing.GpUpU, but not its sequence isomers, UpGpU and UpUpG, was shown to induce Val-sRNA binding to ribosomes.These data indicate that the nucleotide sequence of an RNA codeword for valine is GpUpU.Materials and Methods.-Analyses of poly-and oligonucleotides: (a) Paper electrophoresis was performed on Whatman 54 or 3 MM paper in 0.05 M NH4COOH buffer, pH 2.7, at 80 v/cm for 0.5 hr with authentic reference markers.If a marker was not available, the expected mobility was calculated.7 (b) Descending paper chromatography was performed at room temperature with Whatman 3 MM paper and the following solvents: (A) conc.NH40H-N-propanol-H20, 10/55/35, v/v; (B) 40 gm (NH4)2SO4 dissolved in 100 ml 0.1 M sodium phosphate, pH 7.0.8Bands were visualized under UV light. (c) Ultraviolet absorption measurements were made in a Zeiss spectrophotometer, and spectra were obtained in a Cary recording spectrophotometer The absorbancy of eluates from paper was read against blank paper eluates. (d) The base ratio of the poly UG preparation (D-132) was 0.74/0.26 (U/G).The base ratio of UpUpG, UpGpU, and GpUpU preparations was determined by incubating 2.0 A260 units with 3.5 X 10-3 units of T2 RNase9 in 0.02 ml of 0.5 M ammonium acetate, pH 4.5, for 2.5 hr at 45°.The digestion products were separated by paper electrophoresis, eluted, and the absorbancy of each at appro- priate XmaX was determined.Preparation and separation of oligonucleotides: UpU was characterized as described.6UpUpG and UpG were obtained by incubating 3 X 103 A260 units of poly UG with 1.3 X 103 units of T,- RNase (Sankyo Co., Ltd., Tokyo) in 4.7 ml of 0.1 M NH4HCO3 at 37°for 6 hr.10The reaction mixture was lyophilized, dissolved in 0.1 M (NH4)2 CO3, and terminal phosphates were removed by incubation with E. coli alkaline phosphatase, free of diesterase, as described by Heppel et al." Oligonucleotide fractions were separated by paper chromatography with solvent A for 18 hr.Ultraviolet-absorbing bands with mobilities of UpG and UpUpG were eluted with H20, lyophilyzed, and purified separately by electrophoresis.GpUpU and GpU were obtained by digesting poly UG with purified pork liver nuclease,6 re- moving terminal phosphates, and separating fractions by paper chromatography and electro- phoresis as described above.This procedure yielded a fraction containing UpG and GpU and also one containing GpUpU, UpGpU, and UpUpG.Paper chromatography with solvent B for