An in vivo Approach to tRNA Identity

Author: Normanly, Jennifer

Year: 1989

Degree: Dissertation (Ph.D.)

Advisor: Abelson, John N.

Committee Members: Abelson, John N.; Dervan, Peter B.; Emr, Scott D.; Richards, John H.; Simon, Melvin I.

Option: Biology

DOI: 10.7907/s9xt-6f20

Abstract

A leucine-inserting tRNA has been transformed into a serine-inserting tRNA by changing 12 nucleotides. Only 8 of the 12 changes are required to effect the conversion of the leucine tRNA to serine tRNA identity. The 8 essential changes reside in basepair 11-24 in the D stem, basepairs 3-70, 2-71 and nucleotides 72 and 73, all of the acceptor stem.

Functional amber suppressor tRNA genes were generated for 14 species of tRNA in E. coli, and their amino acid specificities determined. The suppressors can be classified into three groups, based upon their specificities. Class I suppressors, tRNA^Ala2_CUA, tRNA^GlyU_CUA, tRNA^HisA_CUA, tRNA^Lys_CUA, and tRNA^ProH_CUA, inserted the predicted amino acid. The Class II suppressors, tRNA^GluA_CUA, tRNA^GlyT_CUA, and tRNA^Ile1_CUA were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase (AAS). The Class III suppressors, tRNA^Arg_CUA, tRNA^AspM_CUA, tRNA^Ile2_CUA, tRNA^Thr2_CUA, tRNA^Met(m)_CUA and tRNA^Val_CUA inserted predominantly lysine.

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