The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome

Abstract

To unravel the region of human eukaryotic release factor 1 (eRF1) that is close to stop codons within the ribosome, we used mRNAs containing a single photoactivatable 4-thiouridine (s(4)U) residue in the first position of stop or control sense codons. Accurate phasing of these mRNAs onto the ribosome was achieved by the addition of tRNA(Asp). Under these conditions, eRF1 was shown to crosslink exclusively to mRNAs containing a stop or s(4)UGG codon. A procedure that yielded (32)P-labeled eRF1 deprived of the mRNA chain was developed; analysis of the labeled peptides generated after specific cleavage of both wild-type and mutant eRF1s maps the crosslink in the tripeptide KSR (positions 63-65 of human eRF1) and points to K63 located in the conserved NIKS loop as the main crosslinking site. These data directly show the interaction of the N-terminal (N) domain of eRF1 with stop codons within the 40S ribosomal subunit and provide strong support for the positioning of the eRF1 middle (M) domain on the 60S subunit. Thus, the N and M domains mimic the tRNA anticodon and acceptor arms, respectively.

Fig. 1. mRNA containing s⁴U. (A) mRNAs obtained by in vitro transcription in the presence of s⁴UTP (instead of UTP). All 42mer mRNAs contain a GAC codon (underlined) followed by a stop or sense codon (bold). As they differ only by the nature of the latter codon, they were named accordingly. In UGA+1, UGA+2 and UGA+3, the triplet was ‘frameshifted’ relative to the GAC codon by the insertion of one, two or three Gs, respectively. All these mRNAs were used after 5′-end ³²P-labeling. (B) Scheme showing the synthesis of the internally labeled s⁴U*GA mRNA analog. This 42mer mRNA was assembled from two fragments: RNA(1), obtained by in vitro transcription; and RNA(2), synthesized chemically. Ligation was performed in the presence of complementary DNA template and T4 DNA ligase.

Fig. 2. Dependence of crosslink formation on s⁴UGA programmed ribosomes as a function of eRF1 concentration. Autoradiograph of the crosslink pattern obtained after irradiation of 0.1 µM 5′-end ³²P-labeled s⁴UGA mRNA analog with 0.2 µM reassociated ribosomes and with or without 2 µM tRNA^Asp and variable amounts of eRF1. The irradiated mixtures were analyzed by 10% SDS–PAGE. Lanes 1 and 2 are typical of non-phased and phased ribosome–mRNA complexes. Lanes 3 and 4 show the behavior of the phased ribosomes on the addition of different amounts of eRF1. Molecular mass markers are indicated (in kDa).

Fig. 3. Codon dependence of eRF1–mRNA and 19 kDa rP–mRNA crosslink formation. (A) Autoradiograph of the crosslink patterns obtained with stop or sense codons containing 0.1 µM 42mer mRNA analogs in the presence of 0.2 µM reassociated ribosome, 6 µM eRF1 and 2 µM tRNA^Asp (lanes 2–7). For comparison, a control without eRF1 is shown (lane 1). Molecular mass markers are indicated (in kDa) on the left. eRF1–mRNA and 19 kDa rP–mRNA crosslinks are indicated by arrows. (B) Codon-dependent formation of the eRF1–mRNA crosslink. The percentage of this crosslink over all of the crosslinks within the same lane was determined by quantification of the data in (A).

Fig. 4. Crosslink patterns obtained with ‘frameshifted’ mRNA analogs. 5′-end ³²P-labeled frameshifted mRNA (0.1 µM) (Figure 1A) was mixed with 0.2 µM ribosomes and 2 µM tRNA^Asp in the absence or presence of 6 µM eRF1. s⁴UGA mRNA (lanes 1 and 5) was used as a control. Molecular mass markers are indicated (in kDa). The nature of the crosslinks is indicated on the left: (a) >100 kDa rRNA–mRNA, (b) 32 kDa rP–mRNA, (c) 29 kDa rP–mRNA, (d) 19 kDa rP–mRNA and (e) 13 kDa rP–mRNA; the eRF1–mRNA crosslink is marked by an asterisk.

Fig. 5. Crosslink patterns obtained with internally labeled s⁴U*GA mRNA analog and phased ribosomes in the presence of eRF1: lane 1, without nuclease treatment; lane 2, after microccocal digestion of tRNAs. Reaction conditions: 0.1 µM mRNA, 0.2 µM ribosome and, where indicated, 2 µM tRNA^Asp and 6 µM eRF1. Autoradiogram after 12.5% SDS–PAGE. To localize the His₆-tagged eRF1p* product (arrow), an aliquot of the mixture obtained after irradiation of the complete mixture was nuclease treated and passed through an Ni-NTA column. The fraction eluted with 150 mM imidazole was analyzed in parallel (lane 3). Molecular masses of the rPp* and eRF1p* obtained after nuclease digestion are indicated (in kDa) close to the thin arrows.

Fig. 6. Mapping the crosslinking site on wild-type eRF1. (A) Determination of the size of the ³²P-labeled eRF1p* fragments after specific treatments. In lanes 1, 2 and 4, eRF1p* was treated by CNBr (Met↓X), protease V8 (Glu↓X and Asp↓X) and protease Arg-C (Arg↓X), respectively. Samples were analyzed by either 12.5% Tris–glycine (lane 1) or 16.5 % Tris–tricine (lanes 2–5) SDS–PAGE. Digestion of 15 µg of eRF1 with either V8 (lane 3) or Arg-C (lane 5) proteases followed by visualization by silver staining. Asterisks indicate the positions of V8 and Arg-C on the gel. Arrows indicate the position of full-length eRF1. (B) Schematic representation of the sites of cleavage of full-length human eRF1by CNBr or V8 and Arg-C proteases; sites are represented as vertical bars. The size of ³²P-labeled peptides in agreement with the data of (A) are shown in gray (fragments resulting from complete cleavage) or as black bars (incomplete cleavage) for CNBr and V8. The data obtained with Arg-C show that the 9.3 kDa peptide, positions 82–166 (crossed black bar), is not labeled. Compilation of the data is shown as a black box (positions 55–81 of human eRF1).

Fig. 7. Mapping of the crosslink with mutant eRF1s. (A) Patterns of CNBr induced cleavage fragments obtained with S60M, V66M and G73M (left) and S60M, I62M, K63M and S64M (right). Numbers 1–4 and 5–8 refer to fragments shown in (B) and (C), respectively. Samples were analyzed by 12.5% Tris–glycine SDS–PAGE, and wild-type eRF1p* was used as a control. (B) Expected size of ³²P-labeled fragments (shown in gray) for S60M, V66M and G73M, indicating that the crosslink maps in the NIKS region. (C) Size of fragments expected from CNBr induced cleavage of the 52–195 polypeptide on the substitution of S60, I62, K63 or S64 by a Met residue.

Fig. 8. Comparison of the tRNA (A) and eRF1 (B) crystallographic structures. The similarity of the structures is shown by a side view (left) and a front view (right). Regions displaying similar roles (anticodon versus KS and CCA versus GGQ) are colored yellow. Molecular structures are shown by their water accessible surfaces, and the N domain is dark blue. (C) Enlarged view of the N domain of eRF1 showing the relative positions of the site of crosslink (yellow) and of residues E55, S123 and Y125 (orange), previously proposed to contact the base moiety of the invariant U residue of stop codons (Bertram et al., 2000; Muramatsu et al., 2001, Inagaki et al., 2002). Pink sticks show amino acids connecting the N and M domains.