Structure-function relationship in Escherichia coli initiation factors: role of tyrosine residues in ribosomal binding and functional activity of IF-3

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

“…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 phosphate in the absence of 30S ribosomal subunit, became protected in its presence (Ohsawa & Gualerzi, 1981)+ Because the modification of these lysines did not result in IF3 inactivation, however, Arg6 remains the most likely candidate for the establishment of an interaction with the ribosome+ The results of other chemical modifications and mutagenesis also allowed the identification of Cys65, Tyr70, Tyr75, and Lys79 as functionally important, yet perhaps only marginally (or indirectly) involved in binding to the 30S subunits+ The -SH group of Cys65 was modified by various reagents, including fluorescent-and spin-labels+ These experiments indicated that Cys65 is not essential for the biological activity of IF3 but that its rate of modification is slower in 30S-bound IF3 and a nitroxide spin-label at Cys65 became immobilized in titrations with 30S subunits, but not with nucleic acids such as random poly(A,U,G)+ It was suggested that Cys65, which is exposed in free IF3, is located at the edge of the IF3 binding site in the 30S-IF3 complex (Pon et al+ 1982a)+ Like Tyr107, which is located in the "primary RNA binding site" of the molecule (see above), both Tyr residues present in the N-domain (positions 70 and 75) were found to be accessible to lactperoxidase-catalyzed iodination in free IF3 and fully protected in 30S-bound IF3+ Isolated RNA, on the other hand, was found to protect Tyr70 from modification, but not Tyr75+ Furthermore iodination of Tyr70 did not prevent the binding of IF3 to the ribosome, but resulted in the formation of a partially inactive complex, whereas modification of Tyr75 did not produce any detectable inactivation of IF3 in vitro (Bruhns & Gualerzi, 1980)+ A direct influence of Tyr75 on the activity of IF3 in vivo is cl...…”

“…The finding that the C-domain is affected by the addition of the ribosomal subunit more readily and more The coordinates of the structures were obtained from the Protein Data Bank+ The C-domain is the NMR structure of the E. coli protein (PDB entry: 1IFE; Garcia et al+, 1995a) and the N-domain is an energy-minimized structure based upon the backbone coordinates of the X-ray structure of B. Stearothermophilus protein (PDB entry: 1TIG; Biou et al+, 1995), as described in Materials and Methods+ Drawings were made with the program MOLMOL (Koradi et al+, 1996)+ intensely than the N-domain is in full agreement with the localization of the primary RNA binding site of IF3 within this region of the molecule+ Thus, based on the present NMR data and on previous results, three main regions of the C-domain can be singled out as being important for the interaction with the 30S ribosomal subunit+ The first of these regions, spanning from Arg99 through Arg116, is constituted by the distal tip of strand B6, by the loop (L6) connecting strand B6 and helix H3 and by the proximal half of H3+ Nearly all residues in this region are heavily involved in the interaction of IF3 with the 30S subunit+ In fact, this region of the molecule contains Arg99, Gly101, Thr102, Asp103, Gly105, Asp106, Gln108, Lys110, Arg112, Leu114, and Ile115, which were shown to be involved by NMR spectroscopy (Figs+ 4 and 5A)+ Furthermore, other approaches have identified some of the same residues (Arg99, Lys110, and Arg112), as well as additional ones (Tyr107 and Arg116; Fig+ 5B)+ In fact, iodination of Tyr107 (Bruhns & Gualerzi, 1980), modification of Lys110 with pyridoxal phosphate (Ohsawa & Gualerzi, 1981), and amino acid substitutions at the same positions by site-directed mutagenesis (De Bellis et al+, 1992) were found to reduce the association constant of IF3 for the 30S ribosomal subunit severely+ Furthermore, site-directed mutagenesis of Arg99 and Arg116 demonstrated that these residues are also very important for this interaction (Petrelli et al+, 1998; R+ Spurio & C+O+ Gualerzi, unpublished results)+ A functional role for Arg99 is also indicated by the identification of two spontaneous mutants with defective activity, srjA3 and srjA4, in which this residue is replaced by His or Leu, respectively (Haggerty & Lovett, 1993)+ Arg 112, on the other hand, has not yet been mutated or modified in E. coli IF3, but an Arg103 mutant of B. stearothermophilus IF3 (which corresponds to Arg112 of E. coli ) displays reduced binding and reduced activity (Petrelli et al+, 1998; R+ Spurio & C+O+ Gualerzi, unpublished results)+ This region of the molecule probably represents the primary RNA binding site of IF3+ In fact, the NMR spectra show that the residues belonging to this part of the molecule are among the first and most affected by the addition of 30S subunits and mutation and/or chemical modification of at least some of these residues was found to affect directly the binding of IF3 to the 30S subunit whereas both 30S subunit and 16S rRNA were found to protect this region of the molecule from chemical modifications (Bruhns & Gualerzi, 1980;Ohsawa & Gualerzi, 1981)+ The validity of this premise is further strengthened by the structural homologies with other nucleic acid binding proteins (see below)+ The second region of the C-domain that is clearly implicated in t...…”

The Search and Its Outcome: High-Resolution Structures of Ribosomal Particles from Mesophilic, Thermophilic, and Halophilic Bacteria at Various Functional States

“…The ability of IF3 to discriminate noncanonical initiation codons, or to verify codon-anticodon complementarity, has been attributed mainly to IF3N (10).…”

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

“…Their conclusions, which were gleaned from in vitro experiments, were also supported by nuclear magnetic resonance (NMR) spectroscopy (27,28), which in turn showed that most of the IF3 residues interacting with the ribosome were present in the CTD. Every IF3 mutation which led to ribosome-binding defects was mapped to the CTD, while in vivo studies performed with point mutants of IF3 have consistently indicated the role of residues of the NTD in the fidelity function of IF3 (5,11,12,29,30). Further, recent cryo-electron microscopy (cryoEM) studies have placed the NTD of IF3 at the elbow region of i-tRNA while the CTD engages in interactions with the 30S subunit and has a steric clash with H69 during formation of the B2a bridge in the 70S ribosome (8).…”