A Single Mutation in the IF3 N-Terminal Domain Perturbs the Fidelity of Translation Initiation at Three Levels

J. Mol. Biol. (2008) 383, 937–944

doi:10.1016/j.jmb.2008.09.012

Available online at www.sciencedirect.com

COMMUNICATION

A Single Mutation in the IF3 N-Terminal Domain Perturbs the Fidelity of Translation Initiation at Three Levels Dianna Maar 1 , Dionysios Liveris 2 , Jacqueline K. Sussman 1 , Steven Ringquist 3 , Isabella Moll 4 , Nicholas Heredia 1 , Angela Kil 1 , Udo Bläsi 4 , Ira Schwartz 2 and Robert W. Simons 1 ⁎ 1

Department of Microbiology, Immunology, and Molecular Genetics, University of California-Los Angeles, 1602 Molecular Science, Los Angeles, CA 90095, USA 2

Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA 3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3Y75N protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed. © 2008 Elsevier Ltd. All rights reserved.

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Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Vienna, Austria Received 21 May 2008; received in revised form 30 August 2008; accepted 5 September 2008 Available online 16 September 2008 Edited by D. E. Draper

Keywords: translation initiation; IF3; accuracy

*Corresponding author. E-mail address: [email protected]. Present addresses: D. Maar, Department of Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; J. K. Sussman, Department of Microbiology, California State University, Long Beach, CA 90840, USA; S. Ringquist, Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; N. Heredia, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94551, USA. Abbreviations used: IF3, initiation factor 3; NTD, N-terminal domain; CTD, C-terminal domain; SD, Shine–Dalgarno; NIH, National Institutes of Health. 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

IF3 Function at Three Levels

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Introduction Translation initiation factor 3 (IF3) is one of three protein factors required for efficient and specific translation initiation in bacteria1 (for a recent review, see Laursen et al.1). In Escherichia coli, IF3 is an essential 180-residue protein encoded by the infC gene.2 It binds to the small (30S) ribosomal subunit, playing several important roles. As an “antiassociation” factor, it antagonizes the association between the 30S subunit and the large (50S) ribosomal subunit.3 Other extensive in vitro evidence shows that IF3 preferentially dissociates preinitiation complexes containing either noncanonical start codons4,5 or leaderless mRNA,6 and that IF3 selects preinitiation complexes containing the initiator tRNA.7,8 Genetic analysis has shown that mutations in either the N-terminal domain (NTD) or the C-terminal domain (CTD) of IF3 can perturb start-codon discrimination in vivo, suggesting that both IF3 domains are necessary for sensing interactions between the initiator tRNA and the start codon of the mRNA.9,10 Support for this model was provided by genetic studies showing that this discrimination depends on normal basepairing between the codon and the anticodon during initiation.11 CTD mutations also perturb IF3's role in antagonizing initiation on leaderless mRNAs,6 showing that IF3 senses a second key interaction occurring during initiation—that between the 16S rRNA and the mRNA. More recently, O'Connor et al.12 showed that CTD mutations also perturb IF3's role in initiator-tRNA selection in vivo, consistent with earlier in vitro observations by Hartz et al.7 E. coli IF3 functions as a monomer and has a “dumbbell” shape. The CTD has a structural homology with the U1A splicosomal protein and is thought to be the principal domain responsible for 30S subunit binding.13 Thus, all of the effects of CTD mutations could be explained by diminished binding of IF3 to the 30S subunit, which has been directly shown for at least one such mutant protein.9 The NTD does not bind 30S subunits on its own, but appears to contribute to binding stability in the context of the intact IF3 polypeptide.14 Earlier structural and biochemical

studies indicated that only a single IF3 molecule binds to the 30S subunit on opposite sides of the P-sitebound initiator tRNA, with the CTD contacting the platform interface and with the NTD positioned near the E-site.14–16 This model would account for direct inhibition of 30S:50S association, but suggests that IF3 influences initiator-tRNA selection indirectly. In contrast, crystallography studies place the IF3 CTD on the opposite (solvent) side of the 30S platform, where it could not directly prevent 30S:50S association.17 While this discrepancy remains unresolved, a recent cryo-electron-microscopy structure of the entire initiation complex is consistent with the placement of IF3 at the interface.18 The IF3 NTD and CTD are joined by a “linker” that is unstructured in solution but may form an α-helix when bound to the 30S subunit.19 In the model for IF3/30S binding proposed by McCutcheon et al., this interdomain linker spans the cleft between the platform and the head, in the region of the 30S subunit where decoding occurs, and genetic studies show that the linker is critical for IF3 function in vivo.15,20 Petrelli et al. reported that only the CTD is required for the known IF3 functions in vitro, albeit at higher concentrations than required for intact IF3, and they suggested that the NTD and interdomain linker serve only to increase binding affinity.21 However, other evidence20 suggests that the in vivo case may be more complicated. Certain mutations in the linker have no apparent effect on 30S subunit binding in vitro, yet they prevent complementation of an infC deletion and are dominantly lethal to the viable infC19 point mutant allele in vivo. These observations clearly suggest that, in vivo, these particular IF3 linker mutants interact nonproductively with the 30S subunit in a way that blocks the function of the otherwise normal infC19 mutant IF3 (which bears the E134K substitution; see also Table 1). Moreover, de Cock et al. showed that overexpression of the individually engineered NTD and CTD of IF3 also fails to complement an infC deletion, and that the CTD is highly toxic when separately expressed (M. Springer, personal communication).20 Thus, despite the apparent functional capacity of the CTD in vitro, both the

Table 1. Summary of infC mutations infC allele infC1044 infC362 infC263 infC443 infC943 infC561 infC963

Nucleotide changea

Inferred amino acid changeb

Secondary structure elementc

GCT to ACT TAT to TGT TAT to AAT TAT to TCT ACA to TCA GAG to AAG CCA to CGA

A42T Y75N Y75C Y75S T102S E134Kd P162R

α1 (NTD) α2 (NTD) α2 (NTD) α2 (NTD) Loop 6 (CTD) Loop 7 (CTD) Loop 8 (CTD)

a DNA sequence was determined by dideoxynucleotide sequencing of pUC19 plasmids bearing PCR-derived clones of the respective alleles. The primers used in PCR flanked the chromosomal infC gene by several hundred nucleotides to each side, and the entire sequence of each clone was determined. b Amino acid sequences were inferred from the nucleotide sequences. The infC362 mutation was confirmed in the purified protein by amino acid sequencing, accomplished by standard procedures on a cyanogen bromide fragment of IF3 that begins with M68 (automated Edman sequencing required only seven cycles to identify the Asn replacement; data not shown). c See Fig. 1. d The infC561 mutation is identical with, but independent in origin from, the previously isolated infC19 allele.9

IF3 Function at Three Levels

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Fig. 1. Mutations in E. coli IF3. The protein sequence of E. coli IF3 (SWISS-PROT P02999) is shown, with the positions of point mutations described here marked with black arrows, pointing to the mutant residue. Also shown is a consensus sequence of representative bacterial IF3 sequences (excluding Mycoplasma sp.) derived from the Pfam web site at the Sanger Institute. Conservation is indicated by upper-case (100%), lower-case (N 90%), and underlined lower-case (N 80%) letters. Conserved groups included (@) Phe + Tyr, (#) Arg + Lys, (%) Ilv + Leu + Val, ($) Asp + Asn, and (⁎) Ile + Thr. The cylinders and gray arrows indicate the α-helical and β-strand secondary structure elements, as determined by NMR spectroscopy.13,22

linker and the NTD are necessary for IF3 function in vivo. Here, we address the importance of the IF3 NTD in vivo and in vitro by examining the effects of an NTD substitution mutation (infC362; Y75N) on IF3's role in start-codon discrimination, leaderless mRNA translation, and initiator-tRNA selection. We find that this single mutation perturbs all of these aspects of translation initiation fidelity, without substantially altering IF3 binding to the 30S subunit. We discuss these observations within the context of a model in which IF3 “senses” the quality of the preinitiation complex, which it destabilizes whenever any of these fidelity parameters is suboptimal.

The independent isolation of three different mutations at Y75 suggests its key importance in IF3 function, at least in the context of the isolation method used.10,24 Moreover, these alleles were among the most severe alleles isolated (since IF3 is an essential protein, more severe mutations may not be viable). Despite the quite dissimilar nature of the Cys, Asn, and Ser substitutions in these mutants, all have similar hydrogen-bonding capabilities, in common with Tyr. Whether this suggests a specific aspect of IF3 function is not yet clear, but this is currently being tested by further genetic analysis. In this light, it will be particularly informative to know the detailed interactions between the IF3 NTD and the 30S subunit at the atomic level.

Further characterization of viable infC mutants that are defective in IF3 function in vivo

IF3Y75N binds 30S subunits normally

Mutations in the infC gene that perturb the ability of IF3 to discriminate start codons were isolated and partially characterized in a previous study.10 Subsequently, these alleles were recovered by PCR, and their nucleotide sequence was determined. Figure 1 shows these mutations in the context of the E. coli IF3 protein sequence, along with the secondary structural elements of this factor.13,22 A consensus sequence derived from a variety of bacterial IF3 sequences (excluding Mycoplasma sp.) is also shown. The A42T mutation lies in helix α1 of the NTD and changes a residue at or near the hydrophobic core, likely perturbing the folding of this domain. Three mutations (Y75N, Y75C, and Y75S) change a highly conserved tyrosine residue in α2 of the NTD, near the interdomain linker. Importantly, this Tyr residue is near other residues in α1 or in the linker that are thought to interact directly with the 30S subunit.14 The remaining three mutations alter well-conserved residues in the loops of the CTD fold at or near positions thought to interact with the 30S subunit14 and likely, therefore, to perturb binding. The E134K mutation is identical with the previously isolated infC19 allele.9,23

To examine the effect of the Y75N mutation on 30S subunit binding, the pPROEX vector was used to construct and overexpress His-tagged IF3 bearing this mutation (IF3Y75N), as well as the wild-type (IF3WT) and Y107L point-mutant (IF3Y107L) forms, which were affinity purified (free of chromosomally encoded wild-type IF3; not shown), after which the His-tags were removed with TEV protease. Whereas Y75N lies in the NTD, Y107L maps to the CTD and has been shown previously to perturb 30S subunit binding.25 Following incubation of 30S subunits with equimolar amounts of these wild-type or mutant forms, we determined the bound and unbound fractions by sucrose gradient centrifugation and Western blot analysis. Figure 2a shows a typical sedimentation pattern for 30S subunits that corresponds closely with the collected fractions shown in Fig. 2b–d. Under these experimental conditions, essentially all of the IF3WT protein is found in the bound fraction (Fig. 2b), whereas b5% of the IF3Y107L CTD mutant binds, consistent with earlier work.25 In contrast, more than 90% of the IF3Y75N form is bound to the 30S subunit, suggesting that it has, at most, only a quite modest binding defect, which we discuss below.

IF3 Function at Three Levels

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used in vivo homologous recombination to isolate an isogenic plasmid derivative termed “pRS1717-362” (see Table 2), which bears the infC362 mutation (confirmed by sequencing; not shown). These two plasmids were then used to examine the complementation of infC + and infC362 alleles located in the bacterial chromosome (again, as assessed at the level of start-codon discrimination). Lines 1 and 2 of Table 2 show that the infC362 mutation increases the rate of translation initiation from an AUU start codon by about fourfold, relative to an isogenic reporter bearing an AUG start codon (compare differences in the AUU:AUG ratios), as shown previously.10 Importantly, when infC + is present in the low-copynumber plasmid (pRS1717) in an otherwise infC362 cell (line 3), an intermediate phenotype is seen. The same is true for the reciprocal arrangement, where Table 2. Complementation between infC + and infC362

Fig. 2. IF3 binding to the 30S ribosomal subunit. 30S ribosomal subunits were isolated from E. coli by routine methods and incubated with or without equimolar amounts ( 40 pmol) of IF3WT, IF3Y75N, or IF3Y107L, after which they were applied to a 10–30% sucrose gradient and subjected to centrifugation as previously described.26 The IF3WT, IF3Y75N, and IF3Y107L proteins were purified by Histag chromatography and subsequent cleavage of the Histag using TEV protease. (a) Distribution of 30S subunits during sucrose gradient centrifugation. Shown is a typical distribution of 30S ribosomal subunits, as determined with an ISCO UA-6 spectrophotometer. (b–d) Bound and unbound IF3 fractions. To determine the portions of IF3 proteins bound and unbound to 30S subunits, gradient fractions were precipitated, separated by electrophoresis on a 12% Tris–Tricin gel,27 and transferred to a nitrocellulose membrane using standard procedures, and the relative amount of IF3 in each fraction was estimated by Western blot analysis using anti-IF3 antibodies. Visualization of the specific bands was performed using fluorescent-dye-conjugated secondary anti-rabbit antibodies. Fractions are shown such that they correspond to the 30S subunit distribution shown in (a).

infC + and infC362 alleles exhibit codominance in vivo We previously determined that all of the chromosomally located infC mutant alleles listed in Table 1 were recessive to an infC + gene that was present on a high-copy-number (pBR322-based) plasmid, as assessed by effects at the level of start-codon discrimination.10 To better understand the binding studies reported above, as well as other results described below, we undertook a more careful analysis of the dominance relationships between the infC + and the infC362 alleles, under conditions where their gene copy numbers were comparable. To do this, we first cloned the infC + gene into pMAK700, a pSC101based low-copy-number plasmid28 (two to four copies per cell), to create pRS1717. Following that, we

Reporter gene expressionc

Bacterial strainsa Name

Genotype

Plasmid-borne infC alleleb

AUU: AUG

AUUlacZ

AUG-lacZ (×100)d

JK378 JK382 JK378 JK382 JK378 JK382

infC362 infC + infC362 infC + infC362 infC +

None None infC + infC362 infC362 infC +

32 15 27 15 14 14

425 875 810 750 540 830

7.5 1.7 3.3 2.0 2.6 1.7

a Only the relevant genotypes are shown (see Sussman et al.10 for a complete description). b The chloramphenicol-resistant (CmR) low-copy-number plasmids that were temperature-sensitive to replication were pRS1717 (infC +) and pRS1717-362 (infC362). pRS2803, a CmR tetracyclinesensitive derivative of pACYC184,29 was used as noncomplementing control (“none”). pRS1717 was constructed by subcloning the BamHI-HinDIII bearing infC + fragment from pSAΔ1 (kindly supplied by M. Springer) into the HinDIIIBamHI backbone of pMAK700.28 This fragment contains the native infC gene, including its autoregulatory control region, along with portions of the adjacent upstream and downstream native sequences. pRS1717-362 was derived by homologous recombination between the infC + gene in pRS1717 and the infC362 gene in JK378, essentially as described by Hamilton et al.28 Western blot analysis of appropriate haploid strains showed that IF3 expression is no more than ∼ 2-fold higher in the plasmid context, and that the infC362 mutation increases IF3 expression by only ∼ 2-fold, consistent with a predicted decrease in negative autoregulation (data not shown), although we do not know the extent to which autoregulation might be restored in the heterodiploid case. c The AUU-lacZ (λRS536) and AUG-lacZ (λRS537) reporters, in which the translation initiation codons are AUU and AUG, respectively, were present as single-copy λ-prophage-borne lacZ genes and have been described.10 The indicated bacterial strains were transformed with the indicated plasmids by routine methods, selecting for CmR at 30 °C (the permissive temperature for pRS1717 and pRS1717-362 replication), and β-galactosidase levels were determined following growth in LB CmR ( 25 μg/ml) medium as previously described,30 except that cells were grown at 30 °C. Control experiments established that there were insignificant differences in reporter gene expression in cells grown at 30 °C versus cells grown at 37 °C (results not shown). Reporter activity is shown in Miller units. d The AUU:AUG ratio (multiplied by 100) is the ratio of the level of expression from AUU-lacZ reporters to the level of expression from AUG-lacZ reporters in each case, and is taken to represent the rate of translation initiation of the former, relative to that of the latter (see Sussman et al.10).

IF3 Function at Three Levels

the infC362 allele is plasmid-borne (pRS1717-362) and the bacterial chromosome bears the infC + allele (line 4). Since an increase in the AUU:AUG ratio indicates decreased initiation accuracy, and since the AUU:AUG ratio increases only to an intermediate level in each of the infC +/infC362 diploid cases, these alleles are incompletely dominant to one another. This codominance clearly indicates that the product of the infC362 gene (IF3Y75N) interacts productively with the 30S subunit in vivo, competing effectively with the action of wild-type IF3. Importantly, these observations reinforce the conclusion that the infC362 (Y75N) NTD mutation manifests little or no binding defect in vitro or in vivo. Interestingly, when the infC362 allele is present in both locations (line 5), an intermediate effect is also seen, consistent with self-suppression by the infC362 polypeptide at slightly elevated levels of expression (which we have seen in other experimental contexts with this allele and with other alleles; data not shown). A corresponding effect is not seen when infC + is similarly present at both positions (line 6), consistent with the known autoregulation of the infC + gene.9,10,31,32 infC362 mutation increases the translation of leaderless mRNAs Tedin et al. showed that mutations in the CTD of IF3 can perturb the normal ability of IF3 to antagonize translation initiation on leaderless mRNA.6 Since these CTD mutations probably decrease IF3 binding to the 30S subunit, such effects may be indirectly—rather than directly—due to a defect in the ability of IF3 to modulate 30S function. For this reason, we examined two different point mutations at residue 75 [infC362 (Y75N) and infC263 (Y75C)], as well as two mutations in the CTD [infC943 (T102S) and infC963 (P162R)], for their effects on the translation of the leaderless tetR–lacZ mRNA in vivo. Figure 3 shows that all of the mutations tested manifested an increased expression from this reporter fusion, at levels comparable to effects seen with other CTD mutations.6 Importantly, these results show that the IF3Y75N protein is unable to properly antagonize translation initiation on leaderless mRNAs, even though our genetic and biochemical evidence clearly indicate that this mutant form binds essentially normally to the 30S subunit. Thus, it appears that the IF3 NTD plays a direct role in selecting leadered mRNAs for translation initiation, just as it appears to do for start-codon discrimination. infC362 mutation prevents proper tRNA selection in vitro Using the toe-print assay to detect position-specific binding of the 30S ribosomal subunit to mRNA,7,8 it has been established that an important function of IF3 is to select preinitiation complexes (mRNA + tRNA + 30S subunits) in which the initiator tRNAfMet is bound to an AUG initiation codon, over incorrect preinitiation complexes in which an elongator tRNA (e.g.,

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Fig. 3. Effects of infC mutations on the expression of the leaderless tetR–lacZ. Plasmid pRSTetR was transformed into E. coli strain DR599 and its isogenic infC mutant derivatives,10 and β-galactosidase activities (expressed in Miller units) were determined as previously described.30 Standard error bars are shown. Plasmid pRSTetR is a derivative of pRS41430 and was constructed as follows. Plasmid pJOE39733 served as a template for a PCR introducing an EcoRI site upstream of the tetR promoter and a SmaI site after codon 43 of the naturally leaderless tetR gene of Tn1721. The EcoRI-SmaI DNA fragment was then inserted into the corresponding sites of pRS414 yielding pRSTetR, wherein the resulting tetR–lacZ fusion gene is transcribed from its authentic promoter, initiating at A of the AUG start codon. The alleles tested were infC +, infC263 (Y75C), infC362 (Y75N), infC943 (T102S), and infC963 (P162R) (see Fig. 1 for details).

tRNAPhe) is bound to its cognate sense codon (e.g., UUU) located near the AUG initiation codon. Here, we used the same approach to directly examine the effects of the infC362 mutation (Y75N) on initiatortRNA selection. Figure 4a shows a toe-print analysis of 30S ribosomal subunit binding to the phage T4 g32 mRNA, in which the initiation codon (AUG) is followed immediately by a Phe sense codon (UUU). In the presence of the initiator tRNA (uncharged tRNAfMet; lane 1), the 30S toe-print signal corresponds to tRNAfMet bound in the 30S P-site and positioned at its cognate AUG start codon. When uncharged tRNAPhe is also present (at a 10-fold excess of tRNAfMet; lane 2), the predominant toe-print signal is that of tRNAPhe bound at the P-site and positioned at its cognate UUU sense codon (with a faint signal for tRNAfMet binding at AUG). When wild-type IF3 is added at increasing concentrations (5–600 nM; lanes 3–9), binding of tRNAfMet at the AUG start codon is selected over binding of tRNAPhe at the UUU codon, as shown previously7,8 (see Fig. 4 regarding the doublet band at this position). The AUG band is marked with the arrow doublet band under these experimental conditions; tRNAfMet selection is achieved with

IF3 Function at Three Levels

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Fig. 4. Effects of the infC362 mutation (Y75N) on initiator-tRNA selection in vitro. Toe-print analysis was performed as previously described,8 using phage T4 g32 mRNA as template (whose open reading frame begins with AUG.UUU…). 30S subunits were present at 20 nM; uncharged tRNAfMet and tRNAPhe (when present) were present at 500 and 5000 nM, respectively. Toe-print signals corresponding to the AUG (+16 of the gp32 mRNA relative to A of its AUG start codon) and UUU (+19) codons are indicated. The additional band at + 17 is a long-observed feature of toe prints with the gp32 mRNA and is not thought to result from misalignment of the mRNA. Rather, it is apparently due to conformational changes in the 30S subunit that affect the extent to which reverse transcriptase approaches the “toe” of the ribosome in this assay.34 (a) Wild-type IF3 selects tRNAfMet. Lane 1 shows the 30S toe print in the absence of IF3, when tRNAfMet is bound in the P-site at its cognate start codon (AUG). This signal corresponds to a position 15 nt downstream of this AUG. Lane 2 shows the 30S toe print in the presence of tRNAfMet and tRNAPhe (in 10-fold excess), also in the absence of IF3. The predominant toe-print signal corresponds to a position 18 nt downstream of the AUG (or 15 nt downstream of the UUU), and reflects tRNAPhe bound to the P-site at its cognate sense codon (UUU). Lanes 3–9 are the same as lane 2, except that increasing amounts of wild-type IF3 (graduated gray bar) were present (5, 10, 20, 50, 100, 200, and 600 nM IF3 in lanes 3–9, respectively). (b) IF3Y75N is defective for tRNAfMet selection. Toe-print analysis was performed exactly as previously described in (a), except that increasing amounts of IF3Y75N protein were examined (graduated black bar). (c) IF3Y75N interferes with tRNAfMet selection by wild-type IF3. Lanes 1 and 2 were as previously described above. In lanes 3–8, increasing amounts of IF3Y75N (graduated black bar) were added (at 0, 50, 100, 200, 400, and 600 nM, respectively) prior to the addition of wild-type IF3 at 100 nM (gray bar), after which the toeprint reaction was initiated.

50 nM IF3WT (lane 6). In contrast, Fig. 4b shows that tRNAfMet selection is not observed with IF3Y75N, even at a concentration (600 nM) that is 30-fold higher than that of the 30S subunit and, more importantly, 12-fold higher than the level of IF3WT (50 nM) sufficient to achieve full selection. Moreover, Fig. 4c shows that, in the presence of a constant amount of wild-type IF3 (100 nM), increasing amounts of IF3Y75N mutant protein (0.5-fold to 6-fold relative to IF3WT) inhibit proper tRNAfMet selection by IF3WT (lanes 4–8). At the highest IF3Y75N tested (6-fold over wild type; lane 8), tRNAfMet selection is inhibited by N 85% (as determined by densitometric scanning). These results are consistent with recent genetic observations suggesting that mutations in IF3 perturb tRNA selection in vivo,12 although in that work, only mutations that very likely perturb IF3 binding to the 30S subunit were studied. Together, these toe-printing studies show that the IF3Y75N fails to enable proper selection of the correct preinitiation complex (i.e., 30S subunits containing tRNAfMet bound at the AUG start codon of mRNA) and interferes effectively with the ability of wildtype IF3 to do so, consistent with the dominance study described above. It is important to note that while IF3Y107L is also defective for tRNAfMet selection in similar toe-print studies, this defect is fully overcome at an IF3Y107L concentration that is only 5-fold higher than that needed for effective selection by IF3WT.25 This is in sharp contrast to the IF3Y75N case, where no selection is seen even at 12-fold-higher levels despite our demonstration that the 30S subunit binding defect in IF3Y107L is 10–20 times greater than might be the case for IF3Y75N (see above). These toeprint analyses, together with the dominance and binding studies described above, clearly indicate that the modest binding defect of IF3Y75N observed in vitro cannot account for its functional defects in vitro or in vivo. Rather, its effects must be due, almost entirely, to a failure to modulate one or more aspects of 30S function that are involved in the fidelity of translation initiation. Conclusions The most important observation reported here is that a substitution mutation (Y75N) in the IF3 NTD directly perturbs the ability of IF3 to modulate the fidelity of translation initiation at three different levels. Perturbation of start-codon discrimination, as shown earlier,10 likely reflects loss of kinetic discrimination by IF3 against noncanonical start codons, almost certainly at the level of the tRNA/mRNA (codon/anticodon) interaction.11 The Y75N mutation also perturbs the ability of IF3 to antagonize translation initiation on leaderless mRNAs, almost certainly reflecting the absence of the Shine–Dalgarno (SD) sequence and, hence, the quality of the mRNA/16S rRNA interaction.6 Finally, this same mutation directly prevents tRNAfMet selection in vivo, reflecting the specificity of tRNA/30S interaction, very possibly at the level of tRNA/16s rRNA interactions. Importantly, previous genetic work on

IF3 Function at Three Levels

IF3 function at these latter two levels made use of mutations in the IF3 CTD.6,12 Since most or all of these mutations likely decrease IF3 binding to the 30S subunit, their indirect effects on 30S function can be explained on that basis. In contrast, the IF3Y75N protein binds 30S subunits essentially normally, as evidenced by our binding studies and as inferred by its competition with wild-type IF3 in the toe-print studies. Moreover, the infC362 (Y75N) allele exhibits codominance with infC + in vivo. Thus, it appears that this mutant allele binds to the 30S subunit, but is unable to modulate preinitiation complex interactions at three distinct levels: codon/anticodon, SD/ anti-SD, and tRNA/30S subunit. All three of these key interactions within the preinitiation complex occur in the proximity of the 30S site, but are not immediately adjacent to one another.35,36 This makes it difficult to understand how a single amino acid substitution in IF3 can directly contact (and thereby monitor) all three of these interactions. Therefore, we believe that wild-type IF3 modulates a single initiation parameter (the thermodynamic stability of the preinitiation complex), and that the complex is dissociated whenever any of the key interactions of the preinitiation complex is suboptimal. In this view, either an incorrect codon/anticodon pair, the absence (or weakness) of SD/anti-SD pairing, or the binding of an elongator tRNA in the Psite would be suboptimal, thereby rendering the preinitiation complex sensitive to destabilization by IF3. This view is consistent with earlier observations showing that IF3 preferentially destabilizes preinitiation complexes containing tRNAfMet and triplets other than canonical start codons,4,5,7,8 as well as preinitiation complexes formed with leaderless mRNAs.6 We further propose that IF3 mutations such as Y75N are defective in this ability, quite possibly because they fail to properly modulate 30S conformation—a property that has been shown for wild-type IF3.15 The position of the Y75N mutation near the interdomain linker of IF3 may be of particular importance in this regard,14,20 since it is that portion of IF3 that likely spans the flexible distance between the 30S platform and the 30S head. The notion that IF3 acts through an intrinsic property of the 30S subunit is consistent with our recent observation that sublethal concentrations of so-called Psite antibiotics (e.g., kasugamycin and pactamycin), which are thought to inhibit translation initiation, mimic the in vivo effects of infC mutations, including infC362 (D.M., A. Handler, C. Dammel, and R.W.S., in preparation).

Acknowledgements We thank Claudio Gualerzi and Mathias Springer, as well as all members of our laboratories, especially Aaron Handler, for much helpful discussion of this work. We thank Dr. Ellen Weiser for N-terminal analysis of the IF3Y75N mutant protein. D.M

943 and J.K.S. were supported by a National Institutes of Health (NIH) Predoctoral Training Grant (GM07104). N.H. was supported by an NIH Ruth L. Kirschstein National Research Service Award (grant number F31 GM66363). We thank Larry Gold for his support for the toe-print analysis. This work was supported, in part, by grants from the National Science Foundation (MCB-0079305, to R.W.S.), the NIH (GM29265, to I.S.), and the Austrian Science Fund (P12065MOB, to U.B.).

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