The tRNA affinity and specificity of elongation factor *Tu

Elongation factor Tu (EF-Tu) is the bacterial protein responsible for the delivery of aminoacyl-tRNAs (aa-tRNA) to the ribosomal A-site during protein translation. When complexed with GTP, EF-Tu binds to elongator aa-tRNA with high affinity. Interestingly, EF-Tu shows significant binding specificity between the different tRNA bodies. The major focus of this thesis is to determine how the tight binding of elongator aa-tRNAs is achieved and to explore the mechanism by which tRNA specificity is generated. Furthermore, this thesis aims to identify sequence elements in tRNAs which govern its overall binding affinity to EF-Tu.;In order to identify amino acid residues within EF-Tu that are responsible for its high affinity for aa-tRNAs, a set of 20 mutants of Thermus thermophilus EF-Tu were prepared. Each protein contained a single, structurally conservative mutation in the tRNA binding cleft. By measuring the binding affinity of each mutant EF-Tu to four different aa-tRNAs, the amino acid residues which are important in tRNA binding and specificity were identified. A comparison of the mutagenesis data with the available co-crystal structures of EF-Tu bound to two different aa-tRNAs, reveals that thermodynamically relevant contacts are made along the entire tRNA binding cleft including contacts with both the 3' and 5' ends as well as the T-stem of the tRNA. Furthermore, this analysis indicates that the tRNA specificity exhibited by EF-Tu is the result of the variable thermodynamic contributions of a subset of contacts, most of which are made with the tRNA T-stem.;The results of the protein mutagenesis experiments indicated that the 51-63 base pair is a significant contributor to the EF-Tu binding specificity of tRNA. To further explore this, the binding of a series of 51-63 mutant E. coli Leu-tRNALeu to wild type and the Glu390Ala EF-Tu mutant was measured. The results of these experiments show that the presence of a guanosine in the 51-63 base pair significantly stabilizes the binding of tRNA to EF-Tu. A comparison of the tRNA mutagenesis data to the available structures of the EF-Tu˙aa-tRNA complex reveals that the side chain of Glu390 is capable of making a contact with the exocyclic amine group of guanosine at either position 51 or position 63 thereby providing a mechanism by which the 51-63 base pair can contribute to specificity. By determining the variable thermodynamic contributions of different base pairs at 51-63 and using similar data from two other specificity base pairs, 1-72 and 49-65, we are able to present a simple model which permits the binding affinity of most tRNAs to EF-Tu to be predicted. The model provides a means to calculate the binding free energy of any tRNA to EF-Tu based solely on the composition of the 1-72, 49-65 and 51-63 base pairs.