84 Biochimica et Biophysica Acta, 1050 (1990) 84-92 Elsevier BBAEXP 92096 The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex Pascale Romby 1, Hiroshi Wakao 1, Eric Westhof 1, Marianne Grunberg-Manago 2, Bernard Ehresmann 1, Chantal Ehresmann 1 and Jean-Pierre Ebel 1 l lnstitut de Biologie Mol~culaire et Cellulaire du CNRS, Strasbourg and 2 Institut de Biologie Physico-chimique, Paris (France) (Received 16 May 1990) Key words: initiator tRNAfMet; Initiation factor 2; 16S rRNA; Initiation The conformation of the E. coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S. IF2. fMet- ARNt~ "t • AUG • GTP complex has been investigated using several strucOn~specifie probes. As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon ann, the T and D loops and the acceptor ann. Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon ann. In the 308 initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA. Within the 30S subonit, IF2 does not signif~mtly shield defined portions of 16S rRNA, but induces both reduction and enlmncement of reactivity scattered in the entire molecule. Most are constrained in a region corresponding to the deft, the lateral protrusion and the part of the head facing the protrusion. AH the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding. Introduction In Escherichia coli, the initiation of protein synthesis occurs via the formation of a preinitiation complex involving the fMet-ARNtf Met, the 30S subunit, the mes- senger RNA and GTP. Three initiation factors (IF1, IF2, IF3) promote this reaction. IF2 is implicated in fMet-tRNAf Met bin.cling to the ribosomal P site and possesses a ribosome-dependent GTPase activity (for reviews, see Refs. 1-3). Two alternative mechanisms have been proposed, in which the initiator tRNA may bind either to the 30S subunit through a binary complex or to the three factors. 30S ribosomal complex. A com- plex between the fMet-tRNAMf et and IF2 has been detected, but its stability is rather weak [4-6]. The isolated IF2 binds weakly to the 30S subunit, but tightly in the presence of the two other factors [7-9]. Other- wise, both IF2 and IF3 exert allosteric effect on the 30S Abbreviations: IF, initiation factor; El::, elongation factor. Correspondence: P. Romby, Laboratoire de Biochimie, Institut de Biologie Moh~culaire et Cellulaire du CNRS, 15 rue R. Descartes 67084 Strasbourg Cedex, France. ribosomal subunit [9]. Furthermore, it has been pro- posed that IF2 acts on the 30S subunit by kinetically favoring the binding of fMet-tRNAf Met to the 30S sub- unit [10,11]. However, little information is available about the nucleotides of the 16S rRNA and of the initiator tRNA which may interact with IF2 or about possible conformational adjustments induced by IF2 binding. The crystal structure of the E. coil initiator tRNA is known at high resolution [12]. It appears that its tertiary overall structure is similar to that of yeast tRNAphe with, however, some local differences essen- tially located in the anticodon loop and in the acceptor stem. However, the tertiary contacts could not be given with certainty at that resolution or in the absence of refinement: further, no coordinates are presently availa- ble. This paper gives an overview of two different studies designed: (i) to investigate the detailed structure of the initiator tRNA when engaged in the various steps of formation of the 30S initiation complex (amino- acylation, formylation, interaction with IF2 and with the 30S subunit in the presence of the AUG as mes- senger, GTP and IF2) [13]; (ii) to define the 16S rRNA sites that axe shielded or conformationally modified as a 0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)