Conformational change of Escherichia coli initiator methionyl-tRNA(fMet) upon binding to methionyl-tRNA formyl transferase

Abstract

The specific formylation of initiator methionyl-tRNA (Met-tRNA) by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in Escherichia coli. The determinants for formylation are located in the acceptor stem and in the dihydrouridine (D) stem of the initiator tRNA (tRNA(fMet)). Here, we have used ethylation interference analysis to study the interactions between the Met-tRNA(fMet) and MTF in solution. We have identified three clusters of phosphates in the tRNA that, when ethylated, interfere with binding of MTF. Interference due to ethylation of phosphates in the acceptor stem and in the D stem is most likely due to the close proximity of the protein as seen in the crystal structure of the MTF.fMet-tRNA(fMet) complex. The third cluster of phosphates, whose ethylation interferes with binding of MTF, is dispersed along the anticodon stem, which is distal to the sites of tRNA protein contacts. Interestingly, these latter positions correspond to sites of increased cleavages by RNase V1 in RNA footprinting experiments. Together, these results suggest that in addition to the protein, which binds to the substrate tRNA in an induced fit mechanism, the tRNA also undergoes induced structural changes during its binding to MTF.

Figure 1

Cloverleaf structure of E.coli initiator Met-tRNA_N. Nucleotides playing a major role in formylation are shaded dark gray, whereas those playing a minor role are shaded light gray. RNase cleavages that are decreased in the presence of MTF are indicated in red, whereas those that are increased are indicated in green. Arrows, RNase T2 cleavage; arrowheads, RNase V1 cleavage. Figure is reproduced from Ramesh et al. (20).

Figure 2

(A) Autoradiogram of a 15% denaturing polyacrylamide gel of phosphate alkylation experiments with ENU on 5′-[³²P]-labeled Met-tRNA_N using either conditions that preserve the tertiary structure of the tRNA (with Mg²⁺ ions and 20°C) or conditions where the tRNA is denatured (no Mg²⁺ ions and 90°C). Lane OH, alkaline ladder; lane T1, sequencing ladder generated by cleavage with RNase T1. Increasing amounts of ENU (70, 140, 210 and 280 mM) were used for ethylation. Lane 0, incubation control without the addition of ENU. (B) Phosphates that are unreactive under native conditions [indicated by arrows in (A)] are marked by spheres in the three-dimensional wireframe model of the tRNA.

Figure 3

Ethylation interference analysis on MTF.Met-tRNA_N complex formation. The 5′-[³²P]-labeled tRNA was ethylated under native conditions prior to incubation with 0.5 and 0.75 µM MTF. Following native gel electrophoresis to separate unbound and bound tRNA, an alkaline incubation step was used to cleave the tRNA at the ethylated phosphates. An autoradiogram of a 15% denaturing polyacrylamide gel is shown. Lane F, free tRNA (unbound); lane C, tRNA complexed to MTF (bound). Lane U, ethylated 5′-[³²P]-labeled Met-tRNA_N before selection by binding to MTF; lane OH, alkaline ladder; lane T1, sequencing ladder generated by cleavage with RNase T1; lane 0, incubation control without the addition of ENU.

Figure 4

Quantitation of the results obtained from the ethylation interference experiments using (A) Met-tRNA_N or (B) uncharged tRNA_N. The definition of interference is given in the Materials and Methods. The plot represents the average of three independent experiments. Due to spontaneous scission of the tRNA at certain sites, data on some of the phosphate positions are missing: these are p13–p15, p19–p21, p33 and p55–p57. In addition, the extreme ends of the tRNA (p1–p7 and p72–p76) could not be resolved on the gels. Positions where interference is seen in both charged and uncharged tRNA are marked by filled arrowheads; those that essentially show interference with charged tRNA are marked with empty arrowheads on top of the graphs.

Figure 5

X-ray structure of E.coli MTF.fMet-tRNA complex (31) drawn using RASMOL version 2.6 with coordinates obtained from the Protein Data Bank (PDB code 2FMT). MTF is colored gold, the tRNA is in blue wireframe, except for those positions which (A) show decreased (red spacefill) or increased (green spacefill) RNase V1 and T2 cleavage. (B) Same color code for MTF and tRNA, phosphates which show ethylation interference are highlighted in either red spacefill (matching the decreased RNase cleavage) or green spacefill (matching increased RNase cleavage).