Regulatory elements in eIF1A control
the fidelity of start codon selection
by modulating tRNAi
to the ribosome
Adesh K. Saini,1,3Jagpreet S. Nanda,2,3Jon R. Lorsch,2,5and Alan G. Hinnebusch1,4
1Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892, USA;
Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
2Department of Biophysics and Biophysical
eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and
long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in
these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNAi
the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE1)
and SE2, was found to stimulate recruitment of Met-tRNAi
also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required
for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment.
Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI1and SI2, reverse the defects in
TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements
in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding
in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this
conformation to enable full accommodation of Met-tRNAi
Metin the ternary complex (TC) with eIF2?GTP and
Metin the P site for AUG selection.
[Keywords: Translation; initiation; eIF1A; eIF2; initiator; scanning]
Supplemental material is available at http://www.genesdev.org.
Received October 8, 2009; revised version accepted November 20, 2009.
Identification of the translation initiation codon in eu-
karyotes typically occurs by a scanning mechanism in
which the small (40S) ribosomal subunit recruits the
methionyl initiator tRNA (Met-tRNAi
complex (TC) with the GTP-bound form of eIF2 to form
the 43S preinitiation complex (PIC). The 43S PIC then
binds to the mRNA near the m7G-capped 59 end and
scans the leader, using complementarity with the anti-
codon of the initiator as a key means of identifying the
AUG start codon (Pestova et al. 2007). The mechanisms
involved in ribosomal scanning and in distinguishing
AUG from non-AUG triplets by the scanning PIC are
not fully understood.
The factors eIF1 and eIF1A have been shown to
stimulate scanning and assembly of a stable 48S PIC at
AUG, and eIF1 also blocks recognition of near-cognate
Met) in a ternary
triplets, in a reconstituted mammalian system (Pestova
and Kolupaeva 2002). eIF1 appears to act with eIF1A to
promote an open, scanning-conducive conformation of
the PIC (Lomakin et al. 2000; Fekete et al. 2007; Passmore
et al. 2007), and it impedes GTP hydrolysis by the TC in
the absence of perfect base-pairing between the P-site
codon and anticodon of Met-tRNAi
2004). Consistent with this, recognition of AUG elicits
dissociation of eIF1 from the 40S subunit and accelerates
Pi release from eIF2?GDP?Pi in a reconstituted yeast
system (Algire et al. 2005; Maag et al. 2005).
The mechanisms of scanning and AUG selection are
being dissected with genetic tools in budding yeast.
Substitutions in the three subunits of eIF2, eIF5, and
eIF1 were described that increase the frequency of initi-
ation at the UUG start codon of his4-301 mRNA, re-
storing the ability to synthesize histidine (His+pheno-
type) (Yoon and Donahue 1992; Donahue 2000). A subset
of such Sui?(Suppressor of initiation codon mutant)
substitutions affecting eIF1 (encoded by SUI1) appear
to act simply by accelerating eIF1 dissociation from the
PIC (Cheung et al. 2007). Conversely, overexpression of
Met(Unbehaun et al.
3These authors contributed equally to this work.
4E-MAIL firstname.lastname@example.org; FAX (301) 496-6828.
5E-MAIL email@example.com; FAX (410) 955-0637.
GENES & DEVELOPMENT 24:97–110 ISSN 0890-9369/10; www.genesdev.org97
wild-type eIF1 suppresses the Sui?phenotypes of the
SUI5 and SUI3-2 substitutions in eIF5 and eIF2b, re-
spectively (Valasek et al. 2004; Fekete et al. 2007),
conferring the Ssu?(Suppression of Sui?) phenotype.
These findings support the notion that eIF1 is a ‘‘gate-
keeper’’ that impedes start codon selection and whose
inhibitory functions are eliminated at AUG codons, at
least partly, by its dissociation from the 40S subunit.
Presumably, overexpression of eIF1 prevents its release
from the 40S subunit, which would otherwise occur at
a higher frequency at UUGs in Sui?mutants, and thereby
allows scanning to continue downstream.
The eIF1A contains an oligonucleotide/oligosaccharide-
binding (OB) fold domain related to that present in bac-
terial translation initiation factor IF1, but additionally
contains a helical domain comprised of helix a2 and a
310helix connected by a short linker region (Laa), plus
structured N or C strands that pack against different
surfaces of a2. eIF1A also contains long unstructured
N-terminal tail (NTT) or C-terminal tail (CTT) (Fig. 1A;
Battiste et al. 2000). The original genetic selections that
yielded Sui?mutations affecting eIF1, eIF5, or subunits of
eIF2 failed to identify Sui?mutations in TIF11 encoding
yeast eIF1A (Donahue 2000). However, we found pre-
viously that removing the unstructured CTT and the C
strand, Laa, and 310elements of the helical domain by the
C-terminal truncation D108–153 (DC) confers a His+/Sui?
phenotype in the his4-301 background. The DC truncation
also impaired the ability of eIF1A to promote scanning in
a reconstituted mammalian system, leading us to propose
that the C-terminal region of eIF1A acts to enhance
scanning at non-AUG codons, such that its impairment
by DC increases UUG initiation. Remarkably, alanine
substitutions of residues 17–21 (17–21) inthe unstructured
NTT confer an Ssu?phenotype, leading to the proposal
that the NTT acts to inhibit scanning and promote start
codon recognition, such that its inactivation by 17–21
suppresses UUG initiation in Sui?mutants (Fekete et al.
2005, 2007). Thus, the NTT and C-terminal region of
eIF1A appeared to have opposite effects on scanning and
start codon recognition.
Interestingly, the DC truncation, or double-Ala sub-
stitution of eIF1A CTT residues Phe-131 and Phe-133,
also impairs loading of the TC on 40S subunits in PIC
assembly. A well-established manifestation of this defect
is the perturbation of translational control of GCN4
mRNA by four short upstream ORFs (uORFs). Ribosomes
that have translated the 59-most uORF in GCN4 mRNA
(uORF1) and resumed scanning can bypass the remaining
three uORFs (uORF2–uORF4) and reinitiate at the GCN4
AUG when the levels of TC are reduced by eIF2a
phosphorylation by protein kinase Gcn2 in amino acid-
starved cells. The reduced TC concentration enables
a fraction of the reinitiating 40S subunits to rebind TC
only after bypassing uORF2–uORF4, but before reaching
the GCN4 start codon. A similar shift in reinitiation from
uORF2–uORF4 to the GCN4 ORF also occurs in eIF
mutants where the rate of TC loading on 40S subunits is
impaired, constitutively derepressing GCN4 translation
independently of Gcn2 (the Gcd?phenotype) (Hinnebusch
eIF1A. (A) Schematic showing the domains in eIF1A,
indicating the residue number that begins each domain
(above), and locations of the SEs in the CTT sequence
(below). (Constructs a–p) Schematics indicating amino
acids missing (red dashes) in different TIF11 alleles.
(Right) Relative growth, on a scale of 0–10, of his4-301
tif11D strains harboring the indicated TIF11 alleles on
LEU2 plasmids under the conditions described in B.
(B–D) Slg?and His+/Sui?phenotypes of strains harbor-
ing selected TIF11 alleles, described in A, were deter-
mined by spotting serial 10-fold dilutions on synthetic
complete medium lacking leucine (SC-L) supplemented
with 0.3 mM His (+His) or 0.0003 mM His (?His) and
incubating for 3 d (+His) or 6 d (?His) at 30°C.
Mapping the SE elements in the CTT of
Saini et al.
98GENES & DEVELOPMENT
2005). The DC and the F131A,F133A mutations confer
Gcd?phenotypes by this mechanism, and also reduce the
rate of TC loading on 40S subunits in the reconstituted
yeast system (Olsen et al. 2003; Fekete et al. 2005, 2007).
Thus, the eIF1A CTT appears to have dual functions in
promoting TC binding and suppressing UUG initiation,
but it was unclear whether these two functions are
While the F131A,F133A substitution in eIF1A in-
creases the UUG:AUG initiation ratio measured using
HIS4-lacZ reporters differing in these start codons, it does
not confer the His+/Sui?phenotype observed for the DC
truncation, suggesting that other residues removed by DC
besides F131,F133 act to enhance scanning and block
initiation at UUG codons (Fekete et al. 2005). Hence, we
set out to identify all residues in eIF1A that participate in
this key initiation function. In the process, we discovered
that two loosely conserved ;10-residue repeats in the
CTT, dubbed scanning enhancers (SEs), are the critical
elements that both inhibit UUG initiation and promote
TC binding to the 40S subunit, and we provide genetic
and biochemical evidence that these activities are mech-
anistically linked. We further demonstrate that segments
of the helical domain of eIF1A function with the NTT to
impede scanning and promote start codon recognition
and also negatively regulate TC binding. Our results
support a model in which SE elements stabilize a mode
of TC binding that is conducive with scanning but
incompatible with initiation, and thereby block selection
of near-cognate codons, whereas scanning inhibitor (SI)
elements antagonize this mode of TC binding to enable
Identification of partially redundant SE elements
in the eIF1A CTT
The F131A,F133A mutation in the eIF1A CTT increases
the UUG:AUG initiation ratio, but unlike the DC trun-
cation, does not confer a His+/Sui?phenotype in his4-301
cells (Fekete et al. 2005, 2007). To identify the other CTT
residues responsible for the strong Sui?phenotype of
D108–153, we first constructed the set of nested deletions
shown in Figure 1A (constructs b–f) with a common end
point at the predicted N-terminal residue of the unstruc-
tured CTT (Asp-119) (Battiste et al. 2000). The TIF11
alleles on a LEU2 plasmid were introduced into a his4-
301 tif11D ura3 strain harboring wild-type TIF11 on
a URA3 plasmid, and the latter TIF11+plasmid was
evicted by counterselection on medium containing
5-fluoroorotic acid (5-FOA) (Boeke et al. 1987). Western
analysis of whole-cell extracts (WCEs) of these and other
mutants described below showed that none of their
phenotypes could be attributed to reduced eIF1A expres-
sion (Supplemental Fig. S1). The deletion mutants were
tested for His+/Sui?phenotypes by examining cell growth
on (?His) medium containing only 0.1% of the amount of
histidine added to +His medium to fully supplement His?
auxotrophs. Only the largest deletion (D119–133), which
removed F131 and F133, produced a His+phenotype
while also conferring a strong slow-growth (Slg?) pheno-
type on +His medium (Fig. 1A [constructs a–f], B [lanes
a,e,f]). Given that substituting F131 and F133 alone did
not have a His+phenotype (Fekete et al. 2007), we
surmised that residues between 119 and 130 are func-
tionally redundant with F131 and F133. We provisionally
dubbed this hypothetical element Scanning Enhancer 1
(SE1) and that containing F131,F133 SE2, and hypothe-
sized that a strong Sui?phenotype results only when SE1
and SE2are impaired simultaneously.
To support the idea that SE1is functionally redundant
with SE2, we constructed another set of deletions with
a common end point at F133, and thus all lacking
F131,F133 of SE2(Fig. 1A, constructs g–i). Examination of
these mutants revealed that removing residues from F133
to S125 was required to evoke His+and Slg?phenotypes
(Fig. 1B [lanes i vs. h], summary in 1A [constructs g–i]).
Comparing constructs i and h in Figure 1A suggested that
the C-terminal boundary of SE1lies between S125 and
E127. To map the N-terminal boundary of SE1, we deleted
one residue at a time beginning with D119 from construct
D128–133, which lacks F131, F133 of SE2(Fig. 1A, con-
structs j–l). Moderate His+and Slg?phenotypes appeared
only with removal of F121 (Fig. 1C, lanes l vs. k). Together,
these results suggested that SE1maps between F121 and
E127 (Fig. 1A, schematic).
The aforementioned results for constructs e and f in
Figure 1A suggested that F131,F133 occupy the N termi-
nus of SE2. To map the C terminus of SE2, we deleted
residues from the extreme C terminus of eIF1A in a con-
struct where SE1is removed by D119–130 (Fig. 1A, con-
structsm–p).Moderate His+and Slg?phenotypes appeared
only with the deletion of residues 134–136 in construct
p (Fig. 1D, lanes p vs. o), thus suggesting that SE2extends
from F131 to A136 (Fig. 1A, schematic).
To test directly the conclusion that SE1and SE2have
overlapping functions in blocking UUG initiation, we
constructed complete, multiple-Ala substitutions of SE1
or SE2. The SE1* mutation conferred only a slight His+
phenotype (Fig. 2B, lanes a,b) and a correspondingly small
increase in the UUG:AUG initiation ratio measured using
HIS4-lacZ reporters (Fig. 2C, bars a,b). The SE2* mutation,
in contrast, conferred Slg?, a stronger His+phenotype (Fig.
2B,lanes b,c),anda muchlargerincrease intheUUG:AUG
initiation ratio (Fig. 2C, bars b,c). Strikingly, the SE1*,SE2*
double mutation is lethal, preventing eviction of the
TIF11+plasmid (data not shown). However, combining
SE1* with an incomplete substitutionofSE2in which F131
is retained (mutation SE2* + F131) yields a viable mutant
with strong Slg?and His+phenotypes (Fig. 2B, lane d) and
an even larger UUG:AUG initiation ratio than seen for
SE2* alone (Fig. 2C, bars d vs. c). By assaying a pair of
we confirmed that SE1*,SE2* + F131 provokes a larger
increase in the UUG:AUG initiation ratio than does SE2*
or SE1* (Fig. 2E, bars a–d). These findings support the idea
that SE1and SE2are partially redundant elements that
block initiation at UUG, and suggest that SE2is more
crucial than SE1for this function.
tRNA binding by eIF1A affects initiation
GENES & DEVELOPMENT99
Noting that SE1and SE2comprise a loosely conserved
repeat of nine to 10 residues, each containing a pair of Phe
residues (Fig. 2A), we sought to demonstrate that the Phe
pairs are the critical residues in SE1and SE2blocking
UUG recognition. First, we generated constructs com-
bining a complete substitution of one SE element with
an incomplete substitution of the other SE that retains
both Phe residues and compared them with construct
SE1*,SE2* + F131containing only one Phe residue. Both
constructs retaining two Phe residues (Fig. 2B, lanes e,f)
produce weaker Slg?and His+phenotypes (Fig. 2B) and
lower UUG:AUG ratios (Fig. 2C) compared with that
with only a single Phe (Fig. 2C, bar d). Furthermore,
a construct (Fig. 2C, bar g) in which all of the residues in
the two SEs are substituted except for the four Phe
residues produces no Slg?or His+phenotype (Fig. 2B,
lanes g vs. a) and only a slight increase in the UUG:AUG
ratio compared with wild type (Fig. 2C, bars g vs. a). These
results suggest that the number of Phe residues is the
critical determinant of the Slg?and Sui?phenotypes of
Finally, we compared substitutions of only the two
Phe residues in SE1versus those in SE2. F131,133A pro-
vokes a stronger His+phenotype (Figs. 2B, lanes h,i)
and a larger increase in UUG:AUG ratio (Fig. 2D) com-
pared with F121,123A. Although neither double-Phe
substitution confers a Slg?phenotype, the quadruple-
mutant F121,123,131,133A is lethal (data not shown). Thus,
F121,F123 are less critical than F131,133, but become
essential for blocking UUG initiation in the absence of
F131,F133. (The fact that the F131,133A mutant is His+and
Slg+does not contradict our previous findings, as we had
analyzed a Flag-tagged version of this allele. The Flag tag
reduces the His+/Sui?phenotypes and exacerbates the
growth defects of various TIF11 mutations [Fekete et al.
2007].) Together, the results in Figure 2 indicate that the
phenylalanines are the critical residues in SE1and SE2,
which act redundantly to suppress UUG initiation and
support cell growth.
Evidence that elimination of both SEs is lethal owing
to an intolerable defect in scanning or non-AUG
It is possible that precisely substituting both SEs is lethal
because of an intolerable increase in non-AUG initiation.
To test this idea, we asked whether the lethality could
be overcome by the Ssu?mutation 17–21 in the NTT
of eIF1A or by overexpressing eIF1, both of which reduce
UUG initiation in other Sui?mutants. Indeed, 17–21
mutant retains Slg?and His+/Sui?phenotypes (Fig. 3A,
+His and ?His, segments c vs. a) and a high UUG:AUG ra-
tio of 0.5 6 0.05. Importantly, additionally overexpressing
eIF1 from a high-copy (hc) SUI1 plasmid improved the
in blocking UUG initiation. (A) Schematic of eIF1A
showing sequence similarity between SE1 and SE2.
Residues 121–127 were substituted with Ala in
mutant SE1*, and residues 131–135 were similarly
substituted in SE2*. (B) Slg?and His+/Sui?phenotypes
of his4-301 strains containing the indicated TIF11
alleles determined as described in Figure 1B. (C,D)
Strains from B harboring HIS4-lacZ reporter plasmids
with an AUG (p367) or UUG (p391) start codon were
cultured in SC lacking leucine and uracil (SC-LU)
at 30°C, and b-galactosidase activities (nanomoles
of o-nitrophenyl b-D-galactopyranoside cleaved per
minute per milligram) were measured in WCEs.
(E) his4-301 strains with the indicated TIF11 alleles
and harboring the dual luciferase reporter plasmid
pRaugFFuug, containing LUCRenilla and LUCfirefly
coding sequences with AUG or UUG start codons,
and driven by the ADH1 or GPD promoters, respec-
tively, were cultured in SC-LU, and luminescence was
measured in WCEs. The ratio of luminescence, in
relative light units, for the UUG to AUG reporter was
calculated and plotted. Error bars in C–E give the
standard errors of the mean. Differences in bracketed
mean values were judged to be significant by the
Student’s t-test, with P < 0.001 (***).
SE1and SE2elements functionally overlap
Saini et al.
100GENES & DEVELOPMENT
of the SE1*,SE2*,17-21 strain (Fig. 3A, +His and ?His,
segments d vs. c). Similarly, introducing either 17–21,
hc SUI1, or both into the viable mutant SE1*,SE2* + F131
suppressed its His+/Sui?phenotype (Fig. 3A, ?His, seg-
ments f–h vs. e) and (for 17–21) diminished the elevated
UUG:AUG ratio in this viable Sui?mutant (Fig. 2E,
bars e vs. d).
Consistent with the above findings, D117–153, which
removes both SE1and SE2, is also lethal, and this lethality
is suppressed by 17–21 (Fig. 3B, 5-FOA, lanes c vs. d).
Interestingly, D117–153 confers a His+/Sui?phenotype in
viable cells also harboring TIF11+, and this dominant
His+/Sui?phenotype is suppressed by 17–21 or hc SUI1
(Fig. 3B, ?His, lanes c,d and e,f). As shown above for
SE1*,SE2*,17–21, introducing hc SUI1 into the viable
D117–153,17–21 mutant eliminates its residual His+/Sui?
phenotype (Fig. 3C, lanes c vs. d).
The aforementioned results indicate that the lethality
of eliminating both SEs involves an extreme defect in
non-AUG initiation that is lessened by an Ssu?mutation
in eIF1A or overexpressing eIF1. The dominant Sui?
phenotype of D117–153 further indicates that this de-
fective protein can compete with wild-type eIF1A for
incorporation into PICs, but then fails to block UUG
initiation effectively. Given the proposed function of eIF1
in promoting the open, scanning conformation of the
PIC, and the ability of eIF1 overexpression or the 17–21
mutation to suppress the lethality and Sui?phenotypes
caused by eliminating both SE elements, we infer that the
SE elements promote the open, scanning conformation
of the PIC (hence, their designation as SEs). In contrast,
the eIF1A NTT inhibits this open conformation and is
regarded as an SI element, which we dubbed SI1.
Identification of a second SI element in the eIF1A
Our previous finding that deleting residues 108–153 from
eIF1A (DC) confers a Sui?phenotype (Fekete et al. 2005) is
consistent with the elimination of both SE elements, but
overexpression, suppresses the Sui?phenotypes of SE
substitutions. (A) his4-301 strains with the indicated
TIF11 alleles plus hc SUI1 plasmid YEpW-SUI1 or
empty TRP1 vector were streaked on SC lacking
leucine and tryptophan (SC-LW) with 0.3 mM His
(+His) or 0.0003 mM His (?His) and incubated for 3 d
(+His) or 6 d (?His). (B) his4-301 strains containing the
indicated TIF11 alleles or empty LEU2 vector and
harboring TIF11+on a URA3 plasmid (lanes a–f), and
also hc SUI1 or empty TRP1 vector (lanes e,f), were
replica-plated to either SC-LU (lanes a–d) or SC-LUW
(lanes e,f), both containing 0.3 mM His (+His) or
0.0003 mM His (?His), and to either SC-L (lanes
a–d) or SC-LW (lanes e,f), both containing 5.2 mM
5-FOA. Plates were incubated for 3 d (+His), 6 d (?His),
or 8 d (5-FOA). (C) His+/Sui?phenotypes of his4-301
strains containing the indicated TIF11 alleles plus hc
SUI1 or empty vector were determined as in B. (D)
his4-301 strains with the indicated TIF11 alleles,
TIF11+on a TRP1 vector, and the AUG or UUG
HIS4-lacZ reporters were analyzed as in Figure 2C
except that cells were cultured in SC-LUW. (E) his4-
301 strains with the indicated TIF11 alleles, hc SUI1
or empty TRP1 vector, and the AUG or UUG HIS4-
lacZ reporters were analyzed as in D. (F) Schematic of
eIF1A and the extent of C-terminal truncations in the
indicated TIF11 alleles, with a summary of pheno-
types, as in Figure 1A. (G) Slg?and His+/Sui?pheno-
types of his4-301 strains containing the indicated
TIF11 alleles were determined as in Figure 1B. (H)
his4-301 strains with the indicated TIF11 alleles and
the AUG or UUG HIS4-lacZ reporters were analyzed
as in Figure 2C.
SI element substitutions in eIF1A, or eIF1
tRNA binding by eIF1A affects initiation
GENES & DEVELOPMENT101
seems at odds with the lethality observed here for D117–
153 and SE1*,SE2*, which likewise eliminate both SEs .
Because the lethality of the latter mutations is suppressed
by the 17–21 mutation in SI1, we reasoned that D108–153
is viable because it deletes a second SI element (pro-
visionally dubbed SI2) that is located just upstream of the
SEs and would be left intact by the smaller truncation
D117–153. Supporting this interpretation, extending the
deletion in D117–153 by only the single codon for Asn-
116 (producing D116–153) suppressed the lethality of
D117–153 (data not shown). Furthermore, D116–153 con-
fers a smaller increase in the UUG:AUG initiation ratio
than does D117–153 when these alleles are compared in
situations where D117–153 is not lethal, either in the
presence of TIF11+(Fig. 3D) or hc SUI1 (Fig. 3E). D116–
153 also confers weaker Slg?and His+/Sui?phenotypes
than does D117–153 in the presence of hc SUI1 (Supple-
mental Fig. S2A, lanes d vs. e). Thus, it appears that Asn-
116 belongs to the putative SI2and its elimination by
D116–153 suppresses the lethal, hyper-Sui?phenotype of
removing both SEs in D117–153.
To determine whether elimination of N116 fully inac-
tivated SI2, we examined the Sui?phenotypes of con-
structs that extend the deletion of C-terminal domain
(CTD) residues further upstream to also remove P110,
G107, or Q106 (Fig. 3F, constructs c–f). Compared with
D116–153, these larger deletions improved the growth
rate on complete medium and progressively reduced the
Sui?phenotype (Fig. 3G, +His and ?His, lanes d–f vs. c)
and the UUG:AUG initiation ratio (Fig. 3H, bars c–e vs.
b). (The weaker His+phenotype observed for D116–153 vs.
D110–153 likely reflects the relatively poor growth of
the D116–153 mutant evident on +His medium.) Thus,
extending the deletion of CTD residues from N116 to
Q106, removing the entire C strand, 310helix, and linker
Laa (Fig. 3F), constitutes a step-wise reduction in SI2
To confirm this last conclusion and also map the
N-terminal boundary of SI2, we made nested Ala sub-
stitutions from residue 106 to 109 (the last residue of
a2 and Laa linker) in the lethal D117–153 truncation
(Fig. 4A, ‘‘A’’substitutions in constructs c–f). We reasoned
that substitutions encroaching into the N terminus of SI2
should confer an Ssu?phenotype and suppress the lethal,
hyper-Sui?phenotype of D117–153. Indeed, substituting
both Q106 and G107, but not Q106 alone, suppressed
the lethality of D117–153, and the more extensive sub-
stitutions that include residues 108 and 109 conferred
even higher growth rates (Supplemental Fig. S2B, 5-FOA,
lanes c–f vs. b; results summarized in Fig. 4A, +His).
Importantly, the 106–109 substitution (106–109Ala) also
suppressed the lethality of the hyper-Sui?mutation
SE1*,SE2* (data not shown), and the residual His+/Sui?
phenotype of the resulting viable SE1*,SE2*,106–109Ala
mutant was eliminated by extending the Ala substi-
tutions to encompass residues 106–116 in mutant
SE1*,SE2*,106–116Ala (Fig. 4B, ?His, sections c,d). Mu-
tation 106–109Ala also reduced the Slg?and Sui?/His+
against a2, confer an Ssu?phenotype. (A) Mapping
the N-terminal boundary of SI2. Schematic of eIF1A
and the residues lacking (red dashes) or substituted
with alanines (As) in the indicated TIF11 alleles, with
a summary of phenotypes, as in Figure 1A. (B) Slg?and
His+/Sui?phenotypes of his4-301 strains containing
the indicated TIF11 alleles were determined as in
Figure 3A, except the cells were streaked on SC-L
medium, and are summarized on the right. (C,D)
Positions of conserved residues E108P110E111I115N116
in human eIF1A and their probable contacts with the
indicated residues in helix a2 helix (C), and of N strand
residues L26Y28K29and their predicted contacts with
a2 and Laa residues (D). The coordinates of human
eIF1A (PDB:1D7Q) were used to generate structural
models using PyMOL software, showing selected side
chains in stick representation.
Substitutions in region SI2, which packs
Saini et al.
102GENES & DEVELOPMENT
phenotypes of the viable mutant SE1*,SE2* + F131(Fig.
4B, sections h vs. g). Together, the results suggest that
SI2spans residues 107–117 and encompasses Laa, the
310helix, and most of the structured C strand in the
Because all of the components of SI2belong to the
helical domain (Fig. 4C), we asked whether altering
residues in the Laa, 310helix, or C strand that interacts
directly with a2 (Battiste et al. 2000) would also confer an
Ssu?phenotype by disrupting SI2function. We first ex-
amined the effects of Ala substitutions in five such
C strand residues: Glu108, Pro110, Glu111, Ile115, and
Asn116 (Fig. 4C). This mutation (EPEIN-Ala) suppressed
the lethality of the hyper-Sui?allele D117–153 (Supple-
mental Fig. S2B, 5-FOA, lanes b vs. g) as described above
for Ssu?mutation 106–109Ala. Because the structured
N strand of the N-terminal domain (NTD) (residues 26–
33) also packs against a2, we then asked whether
substituting N strand residues Leu26, Tyr28, and Lys29
that are predicted to contact residues in a2 or Laa (Fig.
4D) would likewise confer Ssu?phenotypes. Indeed, this
triple-Ala substitution (LYKAla) suppressed the lethality
of D117–153 (Supplemental Fig. S2B, 5-FOA, lanes b vs. h)
and diminished the elevated UUG:AUG initiation ratio
conferred by D116–153, closely resembling the Ssu?
mutation 17–21 (Fig. 3H, bars f,g vs. b). Consistent with
these findings, the N and C strand mutations LYKAla and
106–109Ala decreased the Slg?and His+/Sui?phenotypes
of the viable mutant SE1*,SE2* + F131(Fig. 5A, sections
a–c vs. d), and, along with 106–116Ala, also reduced the
UUG:AUG ratio in SE1*,SE2* + F131cells (Fig. 5B, bars f–h
vs. e). These results indicate that proper packing of both
N and C strands against a2 is required for the function of
SI2 in supporting UUG initiation in Sui?mutants of
We asked next whether mutations affecting SI2can
suppress the Sui?phenotype of SUI3-2, encoding the
S264Y substitution in eIF2b. Remarkably, 106–116Ala
and LYKAla both diminish the Sui?/His+phenotype of
SUI3-2 cells (Fig. 5C; data not shown) and reduce the
elevated UUG:AUG initiation ratio conferred by SUI3-2
(Fig. 5D), mimicking the Ssu?mutation 17–21 in SI1in
both respects. Interestingly, the DEAR-AASA mutation
described previously (Fekete et al. 2007), which substi-
tutes Asp98, Glu99, and Arg101 of a2 itself and elimi-
nates contacts with C or N strand residues (Fig. 5E), also
confers an Ssu?phenotype in SUI3-2 cells (Fig. 5C,D).
Thus, the integrity of the entire helical domain is required
of SUI3-2. (A) Slg?and His+/Sui?phenotypes of his4-301
strains containing the indicated TIF11 alleles were de-
termined as in Figure 4B. (B) his4-301 strains with the
indicated TIF11 alleles plus the dual luciferase reporter
pRaugFFuug were assayed as described in Figure 2E. (C)
Slg?and His+/Sui?phenotypes of his4-301 strains with
the indicated TIF11 alleles and containing either plasmid
pRSSU13-S264Y-W harboring SUI3-2 (lanes 2–7) or
empty vector (lane 1) were determined as in Figure 1B,
except that the cells were spotted on SC-LW medium.
(D) Strains described in C harboring the AUG or UUG
HIS4-lacZ reporters were analyzed as in Figure 2C. (E)
Predicted interaction of eIF1A residues D98E99A100R101
with C and N strand residues is depicted as in Figure 4C.
SI substitutions suppress the Sui?phenotype
tRNA binding by eIF1A affects initiation
GENES & DEVELOPMENT103
to support the elevated UUG initiation rate in SUI3-2
cells. Finally, eliminating the entire NTT by the DN
mutation confers a stronger Ssu?phenotype compared
with 17–21 (Fig. 5D), indicating that 17–21 only partially
Gcd?phenotypes of Sui?mutations in SE1SE2
are suppressed by Ssu?mutations in SI2
We showed previously that the DC and F131,133A muta-
tions impair TC binding to 40S subunits. When intro-
duced into Flag-tagged eIF1A, these substitutions dere-
press translation of GCN4 mRNA independently of eIF2a
phosphorylation by GCN2. This Gcd?phenotype was
attributed to a reduced rate of TC loading on 40S subunits
that have translated uORF1 and resumed scanning, al-
lowing them to bypass uORF2–uORF4 and reinitiate at
GCN4 without any decrease in TC formation (Olsen et al.
2003; Fekete et al. 2005, 2007). Consistent with these
findings, the SE2* mutation (which eliminates F131,F133)
confers a pronounced Gcd?phenotype, derepressing
by ;10-fold the GCN4-lacZ reporter containing all four
uORFs in nonstarved GCN2 cells (Fig. 6A, Nonstarva-
tion, bars c vs. a). The SE1* mutation confers a smaller
derepression of GCN4-lacZ, and adding to it the Ala
substitutions of all SE2 residues except F131 (mutant
SE1*,SE2*+F131) evokes a stronger derepression than does
SE1* alone (Fig. 6A, Nonstarvation, bars d vs. b). None of
these mutations increases expression of a GCN4-lacZ
construct lacking all four uORFs in nonstarvation condi-
tions (Supplemental Fig. S3A), confirming that they di-
minish the translational repression imposed by the
uORFs. Importantly, co-overexpressing all three eIF2
subunits and tRNAi
hc plasmid (hc TC) reduces the derepression evoked by
the SE1*, SE2*, and SE1*,SE2* + F131mutations (Fig. 6C).
This supports the idea that their Gcd?phenotypes arise
from slower TC loading on reinitiating 40S subunits
scanning downstream from uORF1, which is overcome
by mass action at higher concentrations of TC.
Met, the components of TC, from an
SE substitutions are suppressed by SI substitutions.
(A) GCN2 strains containing the indicated TIF11
alleles and GCN4-lacZ reporter plasmid p180,
depicted at the top, were cultured in repressing
(Nonstarvation) medium (SC-LU) or derepressing
(Starvation) medium (SC-LU lacking isoleucine and
valine and supplemented with 0.5 mg/mL sulfome-
turon), and b-galactosidase activities were assayed in
WCEs. (B) GCN2 strains containing the indicated
TIF11 alleles were analyzed as in A under non-
starvation conditions. (C) GCN2 strains harboring
the indicated TIF11 alleles, GCN4-lacZ reporter
plasmid p180, and either empty vector, hc SUI1, or
hc TC plasmid p4385 were assayed under repressing
conditions as in A, except that cells were grown in
SC-LUW. (D) GCN2 strains harboring the indicated
TIF11 alleles plus the AUG or UUG HIS4-lacZ
reporters and either empty vector, hc SUI1, or hc
TC were analyzed as in Figure 3D. (E) GCN2 strains
harboring the indicated TIF11 alleles, GCN4-lacZ
reporter plasmid p180, and either empty vector or
SUI3-2 were assayed under repressing conditions as
in C. (F) Binding of TC to 40S subunits as a function
of the concentration of 40S subunits (Kd), or time
(kobs), measured as the fraction of [35S]-Met-tRNAi
associated with 40S subunits in a native gel as-
say containing saturating eIF1 (1 mM), model
mRNA(AUG), and wild-type or mutant eIF1A pro-
teins at 1 mM. Errors are mean deviations of at least
three measurements. (G) Effects of eIF1A substitu-
tions on the rate of ribosomal subunit joining.
Kinetics of subunit joining measured using purified
40S and 60S subunits at 100 nM and 200 nM,
respectively, and wild-type or mutant eIF1A proteins
at 1 mM by monitoring the change in intensity of
light scattering over time. Kinetics were biphasic for
wild type, 106–116Ala, and 17–21, but monophasic
for the other three eIF1A mutants. (k1and k2) Rate
constants for the first and second phases, respec-
In vivo and in vitro TC loading defects of
tively; (a1/a2) ratio of amplitudes of the first to second phases; (na) not applicable owing to monophasic reactions. Errors are mean
deviations of at least three measurements.
Saini et al.
104GENES & DEVELOPMENT
The Ssu?mutations 106–109Ala and 106–116Ala in SI2
do not provoke Gcd?phenotypes; rather, 106–116Ala has
the opposite effect of reducing GCN4-lacZ derepression
under starvation conditions, indicating a Gcn?pheno-
type (Fig. 6A, Starvation, bars e,f vs. a). Ssu?mutations in
SI1, including DN, 17–21, and G21D, confer comparable
Gcn?phenotypes (Supplemental Fig. S3B). Importantly,
the Ssu?mutations 106–109Ala, 106–116Ala, 17–21, and
LYKAla all diminish the derepression of GCN4-lacZ
expression conferred by SE1*,SE2*+F131, with 106–
116Ala having the greatest effect (Fig. 6B, bars c–f vs. b).
The Sui?mutation in eIF2b SUI3-2has a Gcd?phenotype
(Williams et al. 1989), and, remarkably, the Ssu?muta-
tions DN, 17–21 and 106–116Ala all greatly suppress the
derepression of GCN4-lacZ in SUI3-2 cells (Fig. 6E).
Together, these findings indicate that the SE and SI
elements have opposite effects on GCN4 translational
control. Whereas SE mutations appear to reduce the rate
of TC loading and confer Gcd?phenotypes, the Gcn?
phenotypes of SI mutations could indicate that they
enhance TC binding to reinitiating 40S subunits scanning
the leader of GCN4 mRNA.
Substituting SE1SE2impairs TC binding in vitro
in a manner suppressed by Ssu?substitutions
in SI elements
To test our prediction that SE and SI substitutions have
opposite effects on TC loading, we measured their effects
on TC binding to 40S subunits in the yeast reconstituted
system (Algire et al. 2002; Acker et al. 2007). We first
examined the effects of SE and SI substitutions on the
equilibrium binding constant of eIF1A for 40S subunits in
the presence of saturating concentrations of wild-type
eIF1, using a previously established assay (Maag and
Lorsch 2003). The results in Supplemental Fig. S4A
indicated that only the two mutants harboring the
LYKAla substitution displayed an appreciable reduction
in eIF1A affinity for 40S subunits. To compensate for
these defects in subsequent TC-binding assays, we used
eIF1 and eIF1A at concentrations of 1 mM, >80-fold above
the Kdvalues measured for all mutant eIF1A proteins.
To assay TC loading, preformed TC containing
40S subunits, a model mRNA with an AUG start codon,
eIF1, and either wild-type or mutant eIF1A, and TC bind-
ing to the 40S subunit was monitored by an electropho-
retic mobility shift (Kolitz et al. 2009). Importantly, the
affinity (greater than eightfold increase in Kd) and rate
(>35-fold decrease in kobs) of TC binding to the PIC (Figs.
6F; Supplemental Fig. S4B). The individual SE1* and SE2*
substitutions also reduced the kobsfor TC loading, but by
smaller amounts—less than twofold and twofold, respec-
SE2* produce moderate and strong Gcd?phenotypes,
respectively (Fig. 6A), whereas SE1*,SE2* is lethal in vivo.
In contrast, the Ssu?substitutions LYKAla, 106–
109Ala, and 106–116Ala all increased the rate of TC
loading in vitro compared with that seen for wild-type
Metwas incubated with purified
eIF1A (Fig. 6F). Remarkably, all three Ssu?substitutions
also greatly reduced the deleterious effects of SE1*,SE2* on
the affinity and rate of TC loading, restoring the Kdvalues
to <0.5 nMand increasingkobsbyfactors of 7–11 compared
with the the SE1*,SE2* mutant alone (Fig. 6F). The SI1
substitution 17–21 also strongly suppressed the defective
rate of TC binding conferred by SE1*,SE2*, although it did
not suppress the reduction in TC-binding affinity (Fig. 6F).
These results suggest that both SI1 and SI2 negatively
regulate the rate of TC loading, and that substitutions in
these regions compensate for the impaired TC binding
conferred by SE substitutions. The fact that SI mutations
cosuppress the TC-binding defects in vitro and the Gcd?
and Sui?phenotypes in vivo of SE mutations strongly
to the rate and stability of TC binding.
Substituting the SE elements stabilizes a closed
conformation of the 40S subunit competent
for subunit joining
Cryo-electron microscopy (cryo-EM) reconstructions of
the 40S subunit in the presence or absence of eIF1 and
eIF1A demonstrated that the two factors synergistically
stabilize the open conformation of the subunit, and bio-
chemical experiments strongly suggested that TC ini-
tially binds to this open state (Passmore et al. 2007).
Accordingly, we hypothesized that the eIF1A SEs might
stimulate the rate of TC loading, at least in part, by
maintaining the open conformation of the 40S subunit.
To test this possibility, we exploited the observation that
eIF1 and eIF1A synergistically impede joining of the 60S
with the 40S subunit, consistent with the idea that the
open conformation is not receptive to subunit joining and
is instead optimized for scanning, whereas subunit join-
ing occurs in the closed conformation following AUG
recognition and eIF1 release (Acker et al. 2006). Hence, if
mutating the SE elements shifts the equilibrium from the
open to closed conformation of the 40S subunit, this
should stimulate the rate of 60S joining with the 40S
subunit in vitro.
Spontaneous joining of the 40S and 60S subunits
follows biphasic kinetics, with the fast and slow phases
having roughly equal amplitudes. These two phases
likely correspond to different conformational states of
the subunit; the fast phase may be 60S joining to the
closed state of the 40S subunit and the slow phase may be
the conversion of the open to closed state of the 40S
subunit (Acker et al. 2006). In the presence of the
SE1*,SE2* mutant, the kinetics of subunit joining were
completely monophasic, with a rate constant (0.05 s?1)
similar to that of the fast phase of joining observed for
wild-type eIF1A (Fig. 6G; Supplemental Fig. S4C). This is
consistent with the idea that substituting the SE ele-
ments shifts the conformational equilibrium of the 40S
subunit in favor of the closed state, which is competent
for subunit joining but not for the initial stage of TC
loading. In contrast, the SI substitutions 17–21 and 106–
116Ala have the opposite effect of increasing the am-
plitude of the slow phase of the subunit joining reaction
tRNA binding by eIF1A affects initiation
GENES & DEVELOPMENT105
(Fig. 6G), which would be expected if they stabilize the
open conformation as a means of enhancing the rate of
TC binding. Strikingly, the SI substitutions 17–21 and
106–116Ala also partially suppress the effect of SE1*,SE2*
by reducing the rate of subunit joining compared with
that given by SE1*,SE2* alone (Fig. 6G; Supplemental Fig.
S4C). The fact that these SI mutations do not fully restore
subunit joining to wild-type behavior is consistent with
the fact that they only partially suppress the TC loading
defects of the SE1*,SE2* substitution. These findings
strongly suggest that the SE and SI elements regulate
the open-to-closed transition of the 40S in opposite di-
rections, which likely contributes to their opposing ef-
fects on the rate of TC loading.
Previously, we provided evidence that the CTTand NTT
of eIF1A differentially regulate the open and closed
conformations of the 40S subunit as the means of
ensuring stringent selection of AUG as a start codon
(Maag et al. 2006; Fekete et al. 2007). Here, we provide
additional evidence for this model, but, importantly, we
also demonstrate that the SE elements in the CTTand SI
elements in the NTT and helical domain regulate scan-
ning and initiation by their differential effects on TC
binding to the 40S subunit. By fine-structure mutational
analysis, we identified a segment in the unstructured
CTTcontaining loosely conserved 10-amino-acid repeats
that act in a partially redundant manner to suppress
initiation at the UUG start codon at his4-301. We dubbed
these repeats SE1and SE2because they appear to promote
the open conformation of the PIC that facilitates contin-
ued scanning when a non-AUG codon occupies the P site.
In-depth analysis revealed that these elements contain
a pair of Phe residues—F121,F123 and F131,F133, respec-
tively—as critical constituents, and that SE2 is more
important than SE1in blocking UUG initiation.
We showed previously that the D108–153 (DC) trunca-
tion of the entire CTT, C strand, 310helix, and part of the
Laa linker, produces a viable Sui?mutant (Fekete et al.
2005). Remarkably, we found here that truncations
smaller than DC that eliminate both SEs without
encroaching extensively into the C strand (e.g., D117–
153) are lethal, as is precisely substituting both SEs by the
SE1*,SE2* mutation. Because the dominant Sui?pheno-
types and lethality of these mutations are suppressed by
overexpressing eIF1 (hc SUI1) or the Ssu?mutation 17–
21, which also suppress the Sui?phenotypes of mutations
in eIF5 (SUI5) and eIF2b (SUI3-2), we can infer that the
lethality of removing both SEs involves an intolerably
high level of initiation at UUG or other near-cognate start
codons that can be mitigated by eIF1 overexpression or
NTT substitutions. Although no TIF11 mutations were
isolated in the original selections that yielded Sui?mu-
tations (Donahue 2000), our results show that substitut-
ing both Phe pairs in the eIF1A SEs increases UUG
initiation comparably with the strongest Sui?mutations
of eIF1, eIF5, or eIF2. Aligning eIF1A sequences from
different species indicates that F121, F131, and F133 are
conserved among lower eukaryotes, but only F121 and
F131 are conserved in animals (Supplemental Fig. S5).
Our results indicate that both F131 and F133 contribute
to the function of SE2(Fig. 2), but F121 might suffice for
The fact that D117–153 is lethal but the more extensive
truncation D108–153 (DC) is viable was the critical clue
that the latter disrupts an element whose elimination
compensates for the lethality of deleting both SEs. By
systematically testing additional mutations for suppres-
sion of the lethality of D117–153, we determined that this
new element includes the Laa, 310helix, and residues
in the structured N and C strands that contact a2 in the
helical domain. Similar to the 17–21 mutation in the
NTT, disrupting the integrity of the helical domain
suppresses Sui?mutations in the SE elements and in
eIF2b (SUI3-2), conferring the Ssu?phenotype. Hence, we
conclude that a2 and its associated N and C strands
comprise a functional unit, dubbed SI2, that acts in
conjunction with SI1to arrest scanning and promote start
Interestingly, mutations affecting SE1 and SE2 also
confer strong Gcd?phenotypes that can be suppressed
by overexpressing TC, indicating reduced rates of TC
loading during reinitiation on GCN4 mRNA. Just as in
blocking UUG initiation, SE2is more critical than SE1in
promoting TC loading and repressing GCN4 translation.
Our biochemical analysis in the reconstituted system
supports this conclusion by revealing reduced rates of TC
binding that are more severe for SE2 versus SE1 sub-
stitutions, and of greatest severity when both elements
are lacking. This last result indicates that SE1and SE2
have overlapping functions in TC loading in addition to
blocking UUG initiation. The strong correlation between
the effects of different SE mutations on the degree of Sui?
phenotypes in vivo and the severity of Gcd?phenotypes
and TC binding defects in vitro provides evidence thatthe
increased UUG initiation in SE mutants is linked mech-
anistically to their defective TC binding.
Remarkably, Ssu?mutations in either SI element di-
minish the Gcd?phenotypes of both SE mutations and
the eIF2b Sui?mutation SUI3-2, thus suggesting that the
Ssu?mutations compensate for defects in TC binding.
Our biochemical data support this conclusion, as defects
in TC binding provoked by the SE1*,SE2* substitution are
partially suppressed by multiple Ssu?substitutions in SI1
or SI2, all of which elicit an increased rate of TC binding
when present in otherwise wild-type eIF1A. Together,
these results provide compelling evidence that the ability
of SI mutations to suppress UUG initiation in Sui?
mutants involves their ability to compensate for TC
loading defects. This leads us to the surprising deduction
that the wild-type SI elements act to oppose continued
scanning and promote start codon recognition by nega-
tively regulating TC binding.
How might the defect in TC binding provoked by SE
mutations lead to increased UUG initiation? We begin by
proposing that the SEs stabilize TC binding to the PIC in
a conformation that is compatible with scanning but
incompatible with initiation. For example, the SEs could
Saini et al.
106GENES & DEVELOPMENT
fully engaged with the P site—a mode of binding we dub
the ‘‘Pout’’ state, which would be associated with the
open, scanning conformation of the PIC (Fig. 7A). The SEs
could promote the Poutmode of TC binding directly (e.g.,
by interacting with the anticodon stem–loop [ASL] of the
initiator), or indirectly by stabilizing the open conforma-
tion of the 40S subunit, to which TC initially binds
(Passmore et al. 2007). In either case, entry of AUG into
the P site and its perfect complementarity with the
anticodon of the initiator would be required to overcome
the SEs and enable Met-tRNAi
dated in the P site and trigger downstream steps in the
initiation pathway. We dub this second conformation
the ‘‘Pin’’state, and envision that it is characteristic of the
closed, scanning-incompatible conformation of the PIC
(Fig. 7A). These two states could be identical to the two
states thatbind TC weakly or stably,respectively, thatwe
detected previously in kinetic studies (Kolitz et al. 2009).
As SE substitutions would impair the ability of eIF1A to
stabilize the Pout conformation in which TC initially
binds, this would account for their deleterious effect on
TC binding to the 40S subunit in vitro and their Gcd?
phenotypes in vivo. By destabilizing Pout, SE mutations
would also facilitate the Pout-to-Pin transition at UUG
codons, which occurs without a perfect codon–anticodon
match, and this would help explain their Sui?phenotypes
TC loading, once TC is bound to the 40S subunit, the
transition from Poutto Pinwould occur more frequently at
near cognate codons. We found that TC overexpression
suppresses the Gcd?phenotypes of SE mutants (Fig. 6C)
but does not reduce the elevated UUG:AUG ratio in
Metin a way that prevents it from being
Metto be fully accommo-
SE1*,SE2* + F131cells (Fig. 6D). These findings are consis-
tent with our model, as increasing TC levels should boost
the rate of TC binding to the Poutstate, reducing the Gcd?
phenotype, but should not mitigate the increased proba-
bility of Pout-to-Pintransitions at UUG codons that, in our
model, contribute to the Sui?phenotypes of SE mutants.
Our proposal that SE elements stabilize TC binding in
a conformation that facilitates scanning but is incompat-
ible with initiation predicts that Met-tRNAi
the P site of the scanning PIC (the Poutstate) in a manner
distinct from that seen in crystal structures of bacterial
70S?mRNA?tRNA complexes (Berk et al. 2006; Korostelev
et al. 2006; Selmer et al. 2006). In fact, this prediction is
strongly supported by results of directed hydroxyl radical
mapping of the mammalian eIF1A in reconstituted 43S
PICs (Yu et al. 2009). This work revealed that the CTT
extends into the P site, threading under the Met-tRNAi
in a configuration that would obstruct Met-tRNAi
binding to the P site in the manner observed in bacterial
70S complexes. Hence, it was concluded that AUG recog-
nition and formation of the closed complex would likely
require removal of the eIF1A CTT from the P site. Indeed,
we argued previously that the eIF1A CTTwould be ejected
from the P site on AUG recognition (Fekete et al. 2007),
based on its physical displacement from eIF1 in the PIC
(Maag et al. 2005) and its AUG-dependent functional
interaction with eIF5 (Maag et al. 2006). It seems likely,
therefore, that the SEs sterically block the Pinmode of TC
binding in addition to stabilizing the Poutconformation
(Fig. 7A). This idea is very attractive because SE mutations
would then facilitate the Pout-to-Pintransition at UUGs
(conferring Sui?phenotypes) in two ways: by destabilizing
Poutand also removing a steric impediment to Pin(Fig. 7B).
tive and negative effects of the SE and SI
elements of eIF1A, respectively, on TC
binding in the Poutconformation, which
is conducive to scanning, and the second
function of the SEs in blocking TC binding
in the Pinconformation, which is incom-
patible with scanning and permissive for
initiation. (B) SE inactivation destabilizes
Pout, reducing TC loading and conferring
the Gcd?phenotype and also enhancing
the Pout-to-Pintransition at UUGs to con-
fer the Sui?phenotype. The Pout-to-Pin
transition and UUG initiation is further
stimulated by loss of the inhibitory effect
of the SEs on Pin. (C) SI inactivation
stabilizes Pout, promoting TC loading and
replacing Gcd?phenotypes with Gcn?
phenotypes, and suppressing the Pout-to-
Pintransition at UUGs to confer the Ssu?
depicting the different conformations of
initiator tRNA in the Pout(D) and Pin(E)
states and the proposed roles of the eIF1A
(A) Model describing the posi-
SE elements in stabilizing initiator binding in Pout, where the initiator is not fully accommodated in the P site, and impeding initiator
binding in Pin, where the initiator is more fully engaged with the P site. On AUG recognition, the SEs are ejected from the P site to
allow greater accommodation of the initiator in the Pinstate.
tRNA binding by eIF1A affects initiation
GENES & DEVELOPMENT107
Combining the results of Yu et al. (2009) with our
finding that SE elements promote TC binding to the
scanning-conducive conformation of the PIC, we propose
that blocks accommodation of the initiator ASL without
preventingthe codon–anticodon interactions required dur-
ing scanning (Fig. 7D). The SEs might interact directly
with the anticodon or ASL of the initiator to stabilize TC
bound in this scanning conformation of the PIC. Pairing
withtheAUG startcodonwould lead toejectionoftheSEs
from the P site and allow more extensive P-site engage-
ment of the initiator ASL in the Pin conformation, as
depicted in Figure 7E. The Pinconformation might re-
semble the ‘‘30S P/I’’ state visualized in a cryo-EM model
of the bacterial 30S initiation complex (Simonetti et al.
2008), and further conformational changes would presum-
ably occur on subunit joining before reaching the classical
P-site binding of tRNA seen in bacterial 70S structures
(Berk et al.2006; Korostelev et al. 2006;Selmer et al. 2006).
Our model in Figure 7, D and E, fits with the notion that
accommodating initiator tRNA in different intermediate
states is a key feature of the small ribosomal subunit
(Simonetti et al. 2008).
We can readily incorporate the functions of eIF1A SI
elements into our model simply by proposing that they
oppose the SEs and destabilize the Poutstate to which TC
mutations partially suppress the defective TC binding
conferred by the SE1*,SE2* mutant in vitro and, consis-
tently, suppress the Gcd?phenotypes of SE mutations in
vivo (Fig. 7C). At the same time, SI mutations would shift
the equilibrium back from Pin to Pout, promoting the
scanning conformation of the PIC and suppressing UUG
initiation in Sui?mutants; i.e., their Ssu?phenotypes (Fig.
7C). The function of the wild-type SI elements in destabi-
lizing the Poutstate can be viewed as a driving force that
facilitates the Pout-to-Pin transition required for AUG
with the P site (Yu et al. 2009), it might play a direct role in
antagonizing initiator binding to the Poutstate (Fig. 7D).
Results of our previous studies suggested that TC binds
initially to the open conformation of the PIC, stabilized
synergistically by eIF1 and eIF1A (Passmore et al. 2007).
Hence, it is plausible that the SEs could stimulate TC
loading by stabilizing the open conformation of the 40S
rather than interacting directly with the initiator. Indeed,
we found that inactivation of the SEs accelerates the rate
of 60S subunit joining, which is thought to proceed only
from the closed conformation of the 40S. Remarkably, SI
substitutions had the opposite effect and partially sup-
pressed the more rapid 60S joining conferred by the SE
substitutions, as would be expected if the wild-type SIs
antagonize the open conformation as the means of pro-
moting start codon recognition. Thus, it appears likely
that the SEs and SIs regulate the rate of TC loading, at
least partly, by their opposing effects on the open-to-
closed conformational transition of the 40S subunit.
However, it is noteworthy in this connection that eIF1
overexpression did not suppress the Gcd?phenotype of
an SE mutation even though it suppressed the Sui?
phenotype (Fig. 6C,D). As eIF1 promotes the open con-
formation, one might expect that overexpressing eIF1
would rescue TC binding in SE mutants if this defect
arises only from reduced occupancy of the open state.
Hence, the SEs likely stimulate TC loading by a second,
possibly direct, mechanism in addition to promoting the
open conformation of the 40S subunit.
In its simplest formulation, our model posits that the
SEs in eIF1A help to recruit TC in the open, scanning
conformation and block initiator accommodation at non-
AUGs, whereas SIs drive the transition to the closed state
in which the initiator is fully engaged in theP site at AUG
codons (Fig. 7). IF1, the bacterial ortholog of eIF1A, lacks
the CTT, NTT, and helical domain (Supplemental Fig. S6;
Carter et al. 2001), whereas the archaeal ortholog (aIF1A)
lacks only the C strand and unstructured CTT (Supple-
mental Figs. S7, S8). Similar to bacteria, it appears that
many mRNAs in archaea use the Shine-Delgarno (SD)
sequence upstream of the start codon to recruit the 30S
subunit directly to the initiation region (Dennis 1997;
Londei 2005), obviating the scanning mechanism. Thus,
in bacteria and archaea, the presence of SD-facilitated
AUG selection is correlated with the absence of SEs,
consistent with the role of these eukaryotic elements in
promoting a scanning-competent intermediate in the
initiation pathway (Fig. 7D). Considering that archaea
resemble eukaryotes in employing a TC with aIF2-GTP
for initiator recruitment (Pedulla et al. 2005), the helical
domains in aIF1A/eIF1A might also play a role in pro-
moting TC binding in the Pin state. Recent in vitro
findings suggest that bacterial IF1 functions in stabilizing
a conformation of the 30S initiation complex that is
incompatible with subunit joining, which can be over-
come by a favorable SD sequence (Milon et al. 2008).
Thus, although IF1 lacks the SE elements, it carries out
one of the functions we ascribe to the SE, of stabilizing
a small subunit conformation incompatible with subunit
joining. This raises the possibility that a region of the OB
fold—the main structural element shared between eIF1A
and IF1—could augment this aspect of SE function.
Materials and methods
Plasmids and yeast strains
Plasmids and yeast strains used in this study are listed in
Supplemental Tables S1 and S2, respectively. Descriptions of
plasmid constructions and site-directed mutagenesis are given in
the Supplemental Material. The his4-301 yeast strain H3582
[MATa ura3-52 trp1D63 leu2-3, leu2-112 his4-301 (ACG) tif11D
p3392 <TIF11, URA3>] was transformed with single-copy or hc
LEU2 plasmids harboring various TIF11 alleles on SC-L medium,
and the resident TIF11+URA3 plasmid p3392 was evicted by
selection on 5-FOA medium to obtain the mutant strains listed
in Supplemental Table S2.
Biochemical assays with yeast extracts
Assays of b-galactosidase activity in WCEs were performed as
described previously (Moehle and Hinnebusch 1991). Measure-
ments of luminescence in WCEs were conducted essentially as
described (Dyer et al. 2000). For Western analysis, WCE extracts
Saini et al.
108GENES & DEVELOPMENT
were prepared by trichloroacetic acid extraction as described
previously (Reid and Schatz 1982), and immunoblot analysis was
conducted as described (Olsen et al. 2003).
Biochemical assays in the reconstituted yeast system
Reagent preparation is described in the Supplemental Material.
Fluorescence anisotropy measurements to determine Kdvalues
for 40S binding of eIF1A were performed as described previously
using wild-type eIF1A Fluorescein-labeled at the C terminus and
competing its 40S binding with unlabeled eIF1A mutants (Maag
and Lorsch 2003; Maag et al. 2006). For all experiments, buffer
conditions were 30 mM HEPES (pH 7.4), 100 mM potassium
acetate (pH 7.4), 3 mM MgCl2, and 2 mM dithiothreitol. TC
binding was measured by gel mobility shift assays as described
previously (Algire et al. 2002). Kinetics of ribosomal subunit
joining in the presence of wild-type or mutant eIF1A was
measured by light scattering on an SX.180MV-R stopped-flow
fluorometer (Applied Photophysics) (Acker et al. 2009).
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