The topographical localization of IF3 on Escherichia coli 30 S ribosomal subunits as a clue to its way of functioning

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy

“…The NMR data obtained in this study and the available amount of biochemical information concerning the interaction of the N-domain with the 30S subunit are definitely less abundant and perhaps not as striking as that obtained for the C-domain+ According to the model proposed (Pon et al+, 1982b), which seems to be supported by the present data, IF3 binds stably to the ribosome through one of its active sites (located in the C-domain) and establishes a fluctuating interaction with the other site (located in the N-domain)+ Thus, the 30S-N-domain interaction, though crucial for the biological activity of IF3, plays a role of secondary importance from the thermodynamic point of view; actually, because it should be easily dissociated, it is not surprising that the N-domain-30S interaction makes use of fewer and less extensive contacts than those of the C-domain+ Furthermore, unlike with the 30S-C-domain interaction, which is predominantly, if not exclusively, an RNAprotein interaction, there are good reasons to believe that protein-protein interactions play an important role in the ribosomal interaction of the N-domain (Pon et al+, 1982a)+ Nonetheless, a number of amino acid residues have been clearly shown to be affected by the 30S subunits in the NMR spectra (Gln8, Ala10, Asn16, Gly17, Glu18, Gln22, Asp52, Ile56, Cys65, Lys72, Phe73, and Ser78) and to be implicated in the functional interaction of IF3 with the ribosome by other criteria (Lys2, Lys5, Arg6, Cys65, Tyr70, Tyr75, and Lys79)+ The N-terminal hexapeptide MKGGKR is the only portion of the N-domain that is known to contribute (directly or indirectly) to the thermodynamic stability of the IF3-30S interaction+ It has been shown that proteolytic cleavage of this peptide substantially reduces the affinity for the 30S subunit as well as the biological FIGURE 6. Model of the complex of initiation factor IF3 with the 30S ribosomal subunit+ A: Overview of the N-and C-domains of IF3 docked to the 16S rRNA in the 30S subunit seen from the solvent side+ B: Close-up of A displaying only the relevant features of the model+ C, D: A Ϫ908 and a ϩ908 rotation around the vertical axis of the image presented in B+ In all panels the 3D model of the 16S rRNA within the 30S ribosomal subunit is that based on EM reconstructions as described in Mueller & Brimacombe (1997) and based on the coordinates provided by the same authors specifically+ The 16S rRNA is shown (in its entirety only in A) completely in black but for the portions to which IF3 has been chemically crosslinked, namely, helices 45 (upper) and 25-26 (lower), which are shown in white+ Other nucleotides indicated are those protected by IF3 from kethoxal (yellow) and CMCT (orange)+ Nucleotide G791, which is partially protected from kethoxal and functionally implicated in IF3 binding by mutagenesis, is indicated in purple+ Nucleotides displaying hyperreactivity in the presence of IF3 to DMS (green) and kethoxal (turquoise) or hypersensitivity to RNase V1 (red) are also indicated+ Further details are found in the text (see Gualerzi & Pon (1990) for a review of these data)+ In all panels a P-site bound tRNA is displayed in magenta in the position indicated by Mueller & Brimacombe (1997)+ IF3 is represented as a blue tube in which the C-terminus of the N-domain and the N-terminus of the C-domain are displayed in darker blue and the residues affected by 30S interaction in yellow (NMR data) or red (mutagenesis or chemical modification data)+ activity of IF3 (Lammi et al+, 1987)+ Furthermore, Lys2 and Lys5, which were exposed to modification with pyridoxal phosph...…”

“…Amino acid residues of IF3 implicated in the interaction with the 30S ribosomal subunit+ The amino acid residues most affected in the experiments by the addition of 30S ribosomes to IF3 and therefore involved in 30S interaction are indicated (in bold) within the primary sequence of IF3+ The identification of the most affected residues was based on a decrease in intensity that was significantly and consistently larger than that of the bulk of the residues+ Underlined residues indicate amino acids that were identified by mutagenesis and/or chemical modification+ Known elements of secondary structure are indicated as reported by Garcia et al+ (1995aGarcia et al+ ( , 1995b+ See text for further details+ unit, thus accounting for the biological effect of IF3 on the association-dissociation of the codon-anticodon interaction in the ribosomal P-site (Pon et al+, 1982b)+ The existence of two separate domains, approximately 46 Å apart (Kycia et al+, 1995), has been fully established by the recent structural studies+ The evidence presented here that the resonances belonging to the amino acids of the C-domain are immobilized by the 30S ribosomal subunit before those belonging to the N-domain (Fig+ 2) clearly indicates that the two domains are not only structurally but also functionally separated in their interaction with the ribosomes+ In fact, the behavior of IF3 in these experiments is compatible with the idea that in the NMR tube there are three species of IF3: free (F-IF3), partly (through the C-domain) bound (PB-IF3), and fully (through both C-domain and N-domain) bound (FB-IF3)+ Upon addition of very low amounts of 30S subunits, the establishment of a medium-fast exchange equilibrium between F-IF3 and PB-IF3 causes the selective broadening of the cross peaks belonging to the C-domain+ Through a first-order isomerization of the initial IF3-30S complex, a portion of PB-IF3 becames FB-IF3; as the concentration of PB-IF3 increases the level of FB-IF3 also increases and the interactions with the N-domain start being detectable through the broadening of the corresponding cross peaks+…”

“…Translational initiation factor IF3 is one of the three proteins required for initiation of protein synthesis in bacteria (for reviews, see Gualerzi & Pon, 1990;McCarthy & Gualerzi, 1990)+ In Escherichia coli, IF3 consists of 180 amino acids encoded by infC, a gene mapped at 37+5 min (Sacerdot et al+, 1982) and proven to be essential for cell survival (Olsson et al+, 1996)+ Acting as a subunit anti-association factor, IF3 supplies the pool of free 30S subunits required for translation initiation (Hershey, 1987, and references therein)+ Furthermore, IF3 accelerates the formation of 30S intiation complexes and ensures the fidelity of translation initiation promoting the dissociation of noncanonical complexes (i+e+, complexes containing aminoacyl-tRNAs other than initiator fMet-tRNA and/or a triplet other than the initiation triplets AUG, GUG, and UUG) (Gualerzi et al+ 1971;Pon & Gualerzi, 1974;Hartz et al+, 1989;Haggerty & Lovett, 1993;La Teana et al+, 1993;Sussman et al+, 1996)+ This function can be accounted for by the influence of IF3 on both on and off rates of codonanticodon interaction in the P-site (Gualerzi et al+, 1977;Wintermeyer & Gualerzi, 1983;Hartz et al+, 1989) and possibly by an IF3-induced adjustment of the mRNA that is shifted from the "stand-by site" to the "P-decoding site" on the 30S ribosomal subunit (La Teana et al+, 1995)+ The interaction of IF3 with the ribosome was one of the first translational functions attributed to the 16S rRNA moiety of the ribosome (for a review, see Santer & Dahlberg, 1996)+ Based on the topographical localization of IF3 on the 30S ribosomal subunit as determined by immunoelectron-microscopy, protein-protein and protein-RNA crosslinking, and the structural and functional properties of IF3 available at that time, Pon et al+ (1982b) suggested that IF3 bridges the cleft of the small ribosomal subunit by binding with two separate sites to both head and platform, and that it fulfills its functional role by affecting the conformational dynamics of the ribosome+…”

The NH2‐terminal cleavage of Escherichia coli translational initiation factor IF3

“…Although bacteriophage MS2 coat protein was found to be the main product in both cases (not shown), a larger amount of short IF3 is needed to stimulate the in vitro translation (fig.4). Upon binding to the 30 S ribosomal subunit, IF3 can be crosslinked by DMS to ribosomal proteins S1 l, $13 and S19 [12]. Since this crosslinking reaction involves lysine residues and since the native to short transition entails the loss of two (out of 20) lysines belonging to what is likely to be a flexible and easily accessible end of the protein [18][19][20], the comparison of the crosslinked products obtained with native and short IF3 could perhaps provide further information on the localization of IF3 on the ribosome by orienting the factor with respect to the ribosomal proteins.…”

“…After 5 rain incubation at 37°C and after centrifugation at 100 000 rpm for 60 min at an actual temperature of -11°C, 75/A of the supernatant were quickly withdrawn for determination of the radioactivity due to unbound IF3. Crosslinking of [3H]NEM-labelled IF3 to 30 S ribosomal subunits with dimethylsuberimidate (DMS) and electrophoretic analysis of the products was carried out as described [12].…”