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

Abstract

'Incubation of CMP in ,H,O with 0.5 M cysteine methyl ester at p2H 5 and 37 "C for 24 h resulted in 43% exchange of 5-H to 5-2H. No deamination of the cytosine nucleus was noted during this treatment. Native and denatured DNA samples from calf thymus were treated in 3H,0 with cysteine methyl ester at pH 5 and 37 "C for 24 h and incorporation of tritium into each 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 z 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.Eschrrichia coli glutamic acid tRNA I1 was treated in 3H20 with 1.24 M cysteine methyl ester at pH 5 and 37 "C. The 24-h-treated tRNA was digested with ribonuclease TI and the fragments were fractionated. Each fragment was then digested with ribonuclease T, into mononucleotides and the radioactivity distribution among the bases was determined. The average radioactivity found for each of the bases of the four major nucleotides was cytosine $ guanine z adenine > uracil.The radioactivity in cytosine varied greatly among the RNase TI fragments, the ratio of the highest to the lowest radioactivity being 18.7. The corresponding value 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 dihydrouridine 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.