Initiation Factor 1 Binds to the Ribosomal Asite

The exact role of IF1 within the initiation complex is perhaps the least understood of the IFs. A number of roles have been described for IF1 including (i) subunit association during 70S initiation complex formation, (ii) modulating the binding and release of IF2 and (iii) blocking the binding of tRNAs to the A-site (reviewed in Refs. [10, 11]). Irrespective the role of IF1, the gene encoding IF1, infA, is essential for cell viability in Escherichia coli [12] indicating its importance in the initiation process.

The structure for IF1 has been determined by NMR spectroscopy and revealed to contain a secondary structure characteristic of the oligomer binding (OB) fold family of proteins, termed because of their ability to bind oligonucleotides and oligosaccharides [13]. The architecture of a classic OB-fold motif includes a five-stranded /2-sheet coiled to form a closed /^-barrel, and capped by a a-helix as exemplified by IF1 (Fig. 7.1-2A). A number of other translational proteins are members of this family including ribosomal proteins S1, S17, and L2 (reviewed in Refs. [14, 15]), tRNA synthetases, IF5A and eIF2a, as well as the central region of eIF1A (Fig. 7.1-2A; [16]). The presence of domains additional to the common OB component in higher organisms, namely archeal and eukaryotic eIF1A, at least partly correlates with the ability to form binary complexes with eIF5B (see following).

Interestingly, a number of the cold shock protein (Csp) family have high structural homology to IF1 (although little sequence homology). In fact, some strains, predominantly Gram-positive, for example Bacillus stearothermophilus, have no obvious IF1 homolog [17]. It has been postulated based on the structural similarity between the Csp and IF1 families that in these strains one of the often many Csps may have assumed this role. In this regard, it is interesting that a double deletion CspB-CspC

Figure 7.1-2 The binding site of IF1 on the 30S subunit and homology with other factors. (A) The solution structures of IF1 (pdblah9; [99]), CspA (pdblmjc; [100]) and eIFIa (pdb 1d7q; [16]), all shown in ribbon representation with strands (dark blue), helices (purple) and random coil (light blue). (B) Overview of the IF1 binding site on the 30S subunit (pdb1hr0; [19]). Ribbon representation of the 16S rRNA (pale blue) including ribosomal proteins (dark blue) with h44 (yellow) and ribosomal protein S12 (green) highlighted. IF1 (purple) is shown as spacefill representation. (C) Close-up view showing that IF1 (purple ribbons) binding, causes A1492 and A1493 (red) to be flipped out of helix 44 (yellow) and the base-pair between A1413 (dark blue) and G1487 (light blue) to be broken.

Figure 7.1-2 The binding site of IF1 on the 30S subunit and homology with other factors. (A) The solution structures of IF1 (pdblah9; [99]), CspA (pdblmjc; [100]) and eIFIa (pdb 1d7q; [16]), all shown in ribbon representation with strands (dark blue), helices (purple) and random coil (light blue). (B) Overview of the IF1 binding site on the 30S subunit (pdb1hr0; [19]). Ribbon representation of the 16S rRNA (pale blue) including ribosomal proteins (dark blue) with h44 (yellow) and ribosomal protein S12 (green) highlighted. IF1 (purple) is shown as spacefill representation. (C) Close-up view showing that IF1 (purple ribbons) binding, causes A1492 and A1493 (red) to be flipped out of helix 44 (yellow) and the base-pair between A1413 (dark blue) and G1487 (light blue) to be broken.

in Bacillus subtilis led to alterations in protein synthesis, cell lysis upon entry into stationary phase, and the inability to sporulate [18]. Deletion of all three Csp proteins was lethal suggesting the importance of having at least one of this family present. Intriguingly, the defects caused by the double knock-out could be cured by the heterologous overexpression of E. coli IF1, suggesting that IF1 could assume some of the chaperone activities normally performed by the Csps. This raises the question if under some conditions the reverse could be true, especially for strains lacking the infA gene. Certainly, there is some evidence that members of the Csp family co-purify with ribosomes; however, this may be related simply to their chaperone activity and reflect their tendency to interact with RNA rather than their involvement in the initiation of protein synthesis.

Despite the low-sequence similarity between OB-fold family members, secondary-structure similarity is striking, as well as the localization of basic residues on one face of the OB-fold. The recent crystal structure of T. thermophilus IF1 bound to the 30S subunit [19] demonstrates that IF1 is no exception. Interactions with the 30S subunit associate with the highly basic surface of IF1, where conserved argin-ine residues (Arg46 and Arg64) stabilize RNA-binding interactions through stacking and electrostatic interactions. The IF1-binding site on the 30S subunit consists of a cleft formed by h44, the 530 loop and protein S12 (Fig. 7.1-2B; [19]). The loop between strands ¡33 and ¡34 is inserted into the minor groove of h44 and flips out residues A1492 and A1493 from their stacked position in h44 (Fig. 7.1-2C). This is reminiscent of the situation where these residues are flipped out due to binding of the antibiotic paromomycin to the decoding site (see Chap. 12) and also due to a cognate tRNA at the A-site (see Chap. 8.2). The major distinction being that during decoding, A1492 and A1493 are critically involved in direct monitoring of correct Watson-Crick pairing of the first two positions of the anticodon-codon duplex [20], whereas within the IF1:30S structure these residues are inaccessible, being protected by IF1 and S12. This suggests that although the binding sites of the A-site tRNA and IF1 overlap, there is little mimicry in their interaction.

Despite the expectation that IF1 would sterically occlude tRNA binding at the Asite [21], it is unlikely that this is the role of IF1 during initiation as there is only one tRNA-binding site on the 30S subunit, that of the prospective P-site [22, 23]; reviewed in Ref. [24]. It seems more probable that IF1 binding at the A-site induces conforma-tional changes that promote subunit association during 70S initiation complex formation. Indeed, the flipping out of A1493 disrupts a base-pair with A1408, destabilizing the top of h44 and allowing lateral movement of bases C1412 and A1413 such that the base-pair between A1413 and G1487 is broken (see Fig. 7.1-2C). This lateral shift moves one strand of h44 with respect to the complementary strand generating "long distance" (up to 70 A from the IF1-binding site) conformational changes within h44 [19]. The minor groove of h44 makes extensive contacts with the 50S subunit, one per helical turn, forming intersubunit bridges B3, B5 and the largest contact point between subunits, bridge B2a [25]. Thus, it is possible that these changes induced by IF1 binding may be responsible for the observed increase in association rates between 30S and 50S subunits [26]. The activation energy associated with 70S formation is large, estimated at 80 kJ moH, and is involved only in adaptation of the 30S subunit (not 50S subunit), rather than the association step itself [27]. Therefore it is tempting to speculate that the initiation factors, particularly IF1 because of the changes it induces in the 30S subunit, help to overcome the free-energy barrier for 50S subunit association with the 30S [28]. The functionally active 30S conformations are obtained by heat activation [29] and have been visualized by cryo-EM, which revealed that they bear a closer resemblance to the 50S-subunit-bound state than to the inactivated state [30]. This heat-activated "intermediate" state may reflect a physiological state [29], such as that induced in vivo by translational factors such as IF1. Indeed, the crystal structure of the IF1-bound 30S subunit [19] also exhibits more similarity to the 50S bound state [25, 31], than to that of the free 30S subunit [32].

Mutation of A1408G eliminates all indicators associated with IF1 binding to the 30S subunit, such as the "tell tale" footprints at A1492 and A1493, yet retains wildtype growth characteristic [33]. This is perplexing as IF1 interaction with the 30S subunit is essential for competent 70S formation [34] and cell survival [12]. The A1408G mutation would also be expected to disrupt the base pair with A1493, tempting speculation that by doing so it enables the 30S subunit to adopt a conformation mimicking that of the initiation complex, thus making IF1 dispensable for cell viability [33]. If this hypothesis would be correct, then direct interaction of IF1 and IF2 may not be necessary and that the 30S conformational change induced by IF1 is sufficient to stimulate IF2 binding.

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