Problems with the transorientation hypothesis.

A novel mechanism for the very first step in polypep-tide elongation on the ribosome has recently been pro-posed by Simonson and Lake (2002)+According to theirtransorientation hypothesis, the incoming aminoacyl-transfer RNA (aa-tRNA) initially binds to the messen-ger RNA (mRNA) with the anticodon stacked on the 59side of the anticodon loop; then, following GTP hydro-lysis, the tRNAswings into theA-site by switching fromthe 59-stacked conformation to the classic 39-stackedgeometry while maintaining Watson–Crick base pair-ing with the mRNA+ There are several serious prob-lems with this proposal+Simonson and Lake inferred the position of theaa-tRNA from immunoelectron microscopy mapping ofthe EF-Tu•GTP•tRNAternary complex on the 30S ribo-somal subunit+ Although this procedure may provide ageneral location for EF-Tu, it is intrinsically incapable ofrevealing the orientation of the incoming tRNA whenthe anticodon first approaches the codon in the 70Sribosome+ Moreover, the binding of the ternary com-plex to the isolated 30S subunit at the A-site in theabsence of the 50S subunit does not occur as part of aphysiological process+ There is, at present, no directexperimental evidence supporting the proposed orien-tation of the EF-Tu•tRNAcomplex at the putative D-sitein the intact ribosome+One major difficulty with the transorientation modelis that it places EF-Tu domain I, which contains theGTPase center, more than 90 A from the a-sarcin-ricin loop (SRL) of the 50S subunit (Fig+ 1)+ TheEF-Tu•GTP•tRNAternarycomplexandEF-Gareknownto have common binding sites on the ribosome, as hasbeen shown by biochemical experiments (Moazed et al+,1988) and by cryo-EM studies (Agrawal et al+, 2000a,2000b)+ These studies have indicated that the GTP-binding domains of both factors lie within5AoftheSRLofthe50Ssubunit+BecausethestructuresofEF-Tuin the cryo-EM study of the kirromycin-stalled 70Sribosome•EF-Tu•GDP•aa-tRNAcomplex(Agrawaletal+,2000a) and the one solved by X-ray crystallography ofthe EF-Tu•GMPPNP•aa-tRNA complex (Nissen et al+,1995) were found to be very similar, and given the factthat kirromycin prevents conformational changes ofEF-Tu immediately after GTP hydrolysis, a large con-formational change between the GTP and GDP statesof the kirromycin-stalled EF-Tu is not expected+ Thetransorientation hypothesis puts EF-Tu in a positionwhere its GTP-binding domain cannot make any con-tact with the SRL (Fig+ 1), even when a very largeconformationalchangeinEF-Tuisallowedtotakeplace+The transorientation model presents serious stericproblems as well+ The authors acknowledge that thereare steric conflicts between EF-Tu and the 50S subunitwhen EF-Tu is docked at the putative D-site+The trans-orientation hypothesis requires L11 and nt 1057–1081of the 23S rRNA to undergo drastic conformationalchanges to permit binding of the ternary complex+Afarsimpler explanation is that EF-Tu binds to the ribosomein the same orientation as seen in cryo-EM reconstruc-tions (Fig+ 1)+ In this orientation, EF-Tu can make con-tact with both L11 and the SRL with no steric conflicts+In transorientation, a steric clash would also occurwhen the aa-tRNA swings from the D-site to the A-site,as it would be forced to sweep through parts of the 50Ssubunit+ In the absence of detailed models for possibleconformational changes in the 50S subunit, it is notpossible to evaluate the claim that transorientation “oc-curs without significant clash +++ between the EF-Tu-