Fig. (5) Crosslinking of endogenous ribosomal protein L36AL in human 80S ribosomes with full length or truncated tRNAox species in the absence or in the presence of the translation termination factor eRF1. (A) Complexes 2: 80S ribosomes incubated in the presence of eRF1 with [5'-32P]tRNAAsp76ox, [5'-32P] tRNAAsp75ox, [5'-32P]tRNAAsp74ox or [5'-32P]tRNAAsp73ox (lanes 1-4, respectively). The controls are: (i) crosslinking in ribosomal complex 1 with [5'-32P]tRNAAsp76ox, in the absence of eRF1 (lane 1*); (ii) crosslinking of [5'- 32P]tRNAAsp76ox with eRF1 in the absence of 80S ribosomes (lane k). (B) 80S ribosomes incubated with [5'-32P]tRNAAsp75ox at pH 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0 (lanes 1-6, respectively) with a free adjacent A-site. Sodium borohydride (NaBH4) was used to trap the fraction of unprotonated reactive ε-amino group that is responsible for the nucleophilic addition to the aldehyde in order to form the Schiff base. (C) Same experiment as in (A) with 80S ribosomes carrying [5'-32P]tRNAAsp76ox at the P-site and eRF1 at the A-site incubated at pH 7.5 and 8.0, respectively (lanes 2) with the following controls. Lanes 1, crosslinking in ribosomal complex 1 with [5'-32P]tRNAAsp76ox, in the absence of eRF1 at pH 7.5 and 8.0, respectively. Lanes k, crosslinking of [5'-32P]tRNAAsp76ox with eRF1 in the absence of 80S ribosomes at pH 7.5 and 8.0, respectively (lanes k). For details see Materials and Methods.