Conformational change of Escherichia coli initiator methionyl-tRNA(fMet) upon binding to methionyl-tRNA formyl transferase.
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.
Mayer
Christine
C
Department of Biology, 68-671A, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
RajBhandary
Uttam L
UL
D000478:Alkylation; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019877:Hydroxymethyl and Formyl Transferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D012358:RNA, Transfer, Met / Q000737:chemistry* / Q000378:metabolism
D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase