Understanding the sequence specificity of tRNA binding to elongation factor Tu using tRNA mutagenesis

Abstract

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and T Psi C stems of Saccharomyces cerevisiae tRNA Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA CAG Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the T Psi C stem primarily account for tRNA binding specificity to EF-Tu.

Figure 1

Sequence of S. cerevisiae tRNA^Phe transcript used in this study with the sites of mutation outlined.

Figure 2

(A.) Measurement of dissociation rates at 0°C in buffer A for: no RNase A (●), Phe-tRNAPhe with (■) and without (▼) EF-Tu●GTP, Phe-tRNA^Phe(G49-U65) (◆), and Phe-tRNA^Phe(A51-U63) (▲). Average k_off values are given in Table I. (B.) NH₄Cl concentration dependence of dissociation rates at 0°C for Phe-tRNA^Phe (●), Phe-tRNA^Phe (G50-U64) (■), Phe-tRNA^Phe (G50-U64 U51-A63) (◆), Phe-tRNA^Phe (G49-U65 C50-G64) (▲). Interpolated values at 0.5 M NH₄Cl (vertical line) are shown in Table I. (C.) Observed rate of association for Phe-tRNA^Phe at 0°C in buffer A at: 0.4 μM (●), 0.2 μM (■), 0.1 μM (◆), 0.05 μM (▲) EF-Tu*GTP. (D.) Association rate dependence on EF-Tu concentration at 0°C in buffer A for: Leu-tRNA^Leu_CAG (●), Phe-tRNA^Phe A49-U65 (■), Phe-tRNA^Phe (◆). Slopes give k_on= 0.08, 0.11 and 0.09 μM^-1s^-1 respectively.

Figure 3

Effects of single base pair substitutions in yeast tRNA^Phe on ΔG° of binding to T. thermophilus EF-Tu in buffer A at 0°C. The solid horizontal line marks the ΔG° for wild-type tRNA^Phe with the sequence given on the x axis, while colored triangles indicate the ΔG° of each mutant at the indicated base pair. Numerical data are shown in Table I.

Figure 4

Free energy of binding to T. thermophilus EF-Tu in buffer A at 0°C of different combinations of strong and weak (outlined) base pairs at the three specificity positions inserted into tRNA^Phe. Numerical data in Table I.

Figure 5

Specificity swap experiment. (A.) Three specificity pairs in tRNA^Leu_CAG were transplanted into tRNA^Phe to give tRNA^Leu/Phe. (B.) Comparison of ΔG° for binding seven Phe-tRNA^X/Phe chimeras to T. thermophilus EF-Tu with the ΔG° for Val-tRNA^X determined previously . The slope of the best fit line is 1.4 with a correlation coefficient of 0.95. Numerical data in Table I.

Figure 6

Additivity test. Comparison of measured ΔG° for double and triple base pair mutants of tRNA^Phe with the value calculated assuming a linear combination of effects found with single base pair mutations. Line indicates perfect correlation between measured and calculated ΔG°.

Figure 7

Comparison of ΔG° values for base pair substitutions in Leu-tRNA^Leu_CAG (numerical data in Table II) and Phe-tRNA^Phe (numerical data in Table I). (A.) Sequence of E. coli tRNA^Leu_CAG with sites of base pair substitutions indicated. (B.) The 49-65 base pair. Line has a slope of 0.82 and a correlation coefficient of 0.93. (C.) The 50-64 base pair. Line has a slope of 1.1 and a correlation coefficient of 0.94. (D.) The 51-63 base pair. Line has a slope of 0.69 and a correlation coefficient of 0.93.

Figure 8

Comparison of previously measured ΔG° of Val-tRNA^X with the calculated ΔG° of the tRNA based on the ΔΔG° values of the 49-65, 50-64, and 51-63 base pairs determined from single base pair mutations. tRNAs are labeled with the one letter code.