Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

Abstract

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3P P-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA Phe , targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3P P-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit. ß

Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome

“…1A) to ribosomal proteins and eRF1 are presented in Figure 2. Cross-linking patterns for analog I have been published earlier (Bulygin et al 2002). Addition of tRNA Phe to nonphased ribosomes with mRNA analogs results in significant enhancement of cross-linking to some ribosomal proteins (Fig.…”

“…lanes 1 and other lanes). Most likely, the protein bands in the upper and middle parts of the gels correspond to cross-linked proteins S2/S3 and S15, respectively, since the locations of these bands conform well to those of the proteins cross-linked to oligoribonucleotides (Bulygin et al 2002;Graifer et al 2004). Strong tRNA-dependent cross-linking to this protein has been shown to be characteristic for perfluorophenyl azide-modified nucleotides in mRNA positions from +4 to +7 (Graifer et al 2004).…”

“…referred to as a ''phased'' ribosome. The irradiated complexes were purified to remove eRF1 cross-linked to mRNA analogs from the ribosome (Bulygin et al 2002).…”

“…However, only mRNA analogs with either s 4 U (Chavatte et al 2001(Chavatte et al , 2002(Chavatte et al , 2003 or perfluorophenylazidemodified uridine at the first stop-codon position or with modified 39 terminal phosphate (Bulygin et al 2002) have been applied so far. These mRNA analogs were able to crosslink to human eRF1 when the modified codon was bound at the A site, and the cross-linking was specific for the mRNA analogs containing a stop or UGG codon (Bulygin et al 2002;Chavatte et al 2002). Since uridine is located in the first position of a stop signal, s 4 U is applicable only for studying surroundings of the first nucleotide of a stop codon.…”

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

“…2). Binding of eRF1 under conditions used here is specific (i.e., occurs only if the A site is occupied with a stop codon [Bulygin et al 2002[Bulygin et al , 2010[Bulygin et al , 2011). Overall, complex 6 mimics a pretermination state, which takes place before GTP hydrolysis, whereas complex 7 corresponds to the post-termination state after GTP hydrolysis.…”