Conformation of Escherichia coli Glutamic Acid tRNA II as Studied by Hydrogen‐Tritium Exchange Catalyzed by Cysteine Methyl Ester

Incubation of CMP in 2 H 2 O with 0.5 M cysteine methyl ester at p 2 H 5 and 37 °C for 24 h resulted in 43% exchange of 5‐H to 5‐ 2 H. No deamination of the cytosine nucleus was noted during this treatment. Native and denatured DNA samples from calf thymus were treated in 3 H 2 O with cysteine methyl ester at pH 5 and 37 ‘C for 24h and incorporation of tritium into Bach DNA base was determined by enzymic digestion of the treated DNA. The order of the specific radioactivity found was cytosine ≫ guanine > adenine ≫ thymine for denatured DNA and guanine > adenine ∼ cytosine > thymine for native DNA. The ratio of radioactivity for denatured/native was 11.6 for cytosine, 1.5 for guanine, 1.8 for adenine and 1.1 for thymine. Hence the incorporation in cytosine under the reaction conditions is preferential for single‐stranded, nonhelical regions of DNA. Escherichia coli glutamic acid tRNA II was treated in 3 H 2 0 with 1.24 M cysteine methyl ester at pH 5 and 37 C. The 24‐h‐treated tRNA was digested with ribonuclease T 1 and the fragments were fractionated. Each fragment was then digested with ribonuclease T 2 into mononucleotides and the radioactivity distribution among the bases was determined. The average radioactivity found for each of the bases of the four major nucieotides was cytosine ∼ guanine ∼ adenine > uracil. The radioactivity in cytosine varied greatly among the RNasc T 1 fragments, the ratio of the highest to the lowest radioactivity boing 18.7. The corresponding valuc for guanine was 11.1, for adenine 4.73 and for uracil 3.64. Based on the data obtained, it was deduced that in this tRNA the anticodon loop, the dihydrouridinc loop and the extra loop were ‘exposed’ under the conditions employed for the labeling. The 5′‐terminal cytosine of the anticodon loop was in a ‘non‐exposed’ state, a situation similar to that previously reported for E. coli tyrosine tRNA [Cashmore, A. R., Brown, D. M. & Smith, J. D. (1971) J. Mol. Biol. 59, 359‐373] and for E. coli formylmethionine tRNA [Goddard, J. P. & Schulman, L. H. (1972) J. Biol. Chem. 247, 3864‐3867]. Both cytosine 48, located at the 3′‐terminal of the extra loop, and guanine 15 in the dihydrouridine loop were in an ‘exposed’ state. This finding does not agree with a tRNA model in which this pair of cytosine and guanine, commonly found in tRNA sequences, forms hydrogen bondings. Positions 30–32, 61–64 and 71, which are located in the stems, were found to be strongly ‘buried’