BACKGROUND
translation of most mrnas in eukaryotic cells occurs by a scanning mechanism wherein the small  ribosomal subunit recruits methionyl initiator trna  in a ternary complex with eif2-gtp, in a reaction stimulated by other eifs, and the resulting 43s pre-initiation complex  binds near the m <dig> g-cap structure of the mrna to assemble the 48s pic.
attachment of the 43s complex at the mrna 5' end is stimulated by the eif4f complex, comprised of cap-binding protein eif4e, the scaffold subunit eif4g, and the dexd/h-box helicase eif4a, which is thought to provide a single-stranded region in the mrna for recruiting the ribosome.
binding sites in eif4g for either eif <dig>  or eif <dig> and eif <dig>  are thought to facilitate recruitment of the 43s pic to eif4f bound at the cap structure.
eif4g also harbors a binding site for the poly-binding protein  that, together with an rna binding domain in the middle region of mammalian eif4g, increases the stability of eif4f binding to the mrna 5' end and also mediates circularization of mrna in the activated eif4f·mrna·pabp mrnp  <cit> .
in addition to stimulating recruitment of the 43s pic to the mrna 5' end, there is evidence that the atp-dependent rna helicase activity of eif4a facilitates ribosomal scanning through secondary structures in the 5' utr to enhance recognition of the aug start codon  <cit> .
however, other dexd/h helicases have been implicated in scanning through long or structured 5' utrs, including ded1/ddx <dig> in yeast  <cit>  and dhx <dig> in mammals  <cit> , and it is uncertain whether eif4a and its binding partners in eif4f are critically required for scanning.
in fact, 43s recruitment and location of the start codon has been reconstituted in vitro for an artificial mrna with an unstructured 5' utr in the absence of eif4f, eif4a, eif4b, and atp, requiring only the eif2·gtp·met-trnai met ternary complex, eif <dig>  eif <dig>  and eif1a  <cit> .
hence, it is possible that native mrnas devoid of stable structures in the 5'utr could be translated at relatively high efficiencies in the absence of eif4f.
indeed, we showed previously that genetically depleting eif4g from yeast cells reduces general translation initiation but does not impair 48s pic formation in vivo by two native mrnas   <cit> .
based on its presumed functions in mrna activation and scanning, it is generally assumed that eif4f plays an important role in determining the relative efficiencies of translation among the repertoire of cellular mrnas and, hence, is a key factor in translational control of gene expression  <cit> .
we examined this hypothesis in yeast by measuring the effect of genetically depleting eif4g from yeast cells on translational efficiencies of mrnas genome-wide.
the depletion of eif4g was very effective and it reduced protein synthesis rates by a factor of ~ <dig>  leading to cell growth arrest.
surprisingly, however, the translational efficiencies of most mrnas were not substantially affected by eif4g depletion.
an intriguing consequence of a strong reduction in eif4g levels was to narrow the range of translational efficiencies genome-wide by reducing the translation of many mrnas with higher than average translational efficiencies in wild-type cells while increasing the translation of different mrnas that are normally translated with lower than average efficiencies.
our findings suggest that eif4g is not essential for translation of any mrnas in yeast cells, but it enhances the differentiation of translational efficiencies among cellular mrnas.
RESULTS
depletion of eif4g <dig> in cells lacking eif4g <dig> evokes a marked decrease in the rate of translation initiation in vivo
to examine the consequences for global translation of eliminating both isoforms of eif4g, we employed a strain deleted of the chromosomal gene encoding eif4g <dig>  and harboring a temperature-sensitive degron allele  <cit>  of the gene encoding eif4g <dig> .
the tif4631-td allele encodes ubiquitin and a thermolabile dihydrofolate reductase moiety fused to the n terminus of eif4g <dig>  expressed from a copper-dependent promoter, and is integrated into the chromosome in a manner that disrupts the resident wild-type  tif <dig> allele  <cit> .
the strain also contains a galactose-inducible form of the gene encoding the ubiquitin ligase  required for proteasomal degradation of degron-tagged proteins by the "n-end rule" pathway  <cit> .
shifting cells from medium containing copper and raffinose  at 25°c to medium containing galactose and raffinose but lacking copper at 36°c represses new synthesis and triggers proteasomal degradation of the existing degron-tagged eif4g1-td protein.
we showed previously that under non-permissive conditions this degron mutant cannot form colonies from single cells, exhibits a strong reduction in doubling time within  <dig> h, and essentially ceases growth and division by  <dig> h after the shift to non-permissive conditions.
this growth arrest can be reversed by shifting cells back to permissive conditions  <cit> .
consistent with our previous results, incubation for  <dig> h under non-permissive conditions was required to deplete eif4g1-td in whole cell extracts  below the detection limit of western analysis .
note that both the wild-type  and mutant wces appear to contain an n-terminally truncated form of eif4g <dig> that migrates more rapidly than either the wt or degron-tagged full-length proteins .
because this truncation is subject to degradation in the degron mutant, but necessarily lacks the n-terminal modifications necessary for n-end rule degradation, it is likely generated from the full-length proteins in vitro following cell lysis.
after  <dig> h of depletion, the degron mutant exhibits the expected reduction in total polysomes and commensurate increase in 80s monosomes, leading to a decreased ratio of polysomes to monosomes  by a factor >  <dig> compared to the p/m ratio for the wt strain under the same conditions .
this is the stereotypical consequence of selective impairment of translation initiation, involving a decrease in new initiation events, "run-off" of elongating ribosomes from existing polysomes, and subsequent accumulation of excess free subunits as 80s couples.
note that depletion of two essential subunits of the eif <dig> complex, in a separate mutant expressing degron-tagged forms of these proteins  <cit> , evokes a more complete polysome run-off than observed in the eif4g1-td mutant .
consistent with the polysome profiles, the rate of total protein synthesis, measured by incorporation of radioactive methionine into acid-insoluble material, was reduced in the eif4g1-td mutant to ~30% of the wt value  after  <dig> h in the non-permissive condition, whereas the eif <dig> degron mutant displayed no detectable -met incorporation under these conditions .
thus, in accordance with our previous conclusions, depletion of eif4g <dig> in cells lacking eif4g <dig> leads to a marked reduction in the rate of translation initiation, but one less severe than that provoked by a comparable depletion of eif <dig> subunits  <cit> .
depletion of eif4g narrows the range of mrna translational efficiencies genome-wide
although a significant level of translation continues following the extensive depletion of eif4g in the degron mutant , it was possible that translation of some mrnas would be greatly diminished while translation of others would continue relatively unaffected or even increase.
to address this possibility, we determined the effect of depleting eif4g on the translational efficiencies of mrnas genome-wide.
to this end, we conducted microarray analysis on rna isolated from the "heaviest" polysomes, containing  <dig> or more elongating 80s ribosomes per mrna , and also total rna from wces, from both degron mutant and wt cells cultured for  <dig> h under non-permissive conditions.
translational efficiencies  were calculated for each gene as the ratio of hybridization intensities on microarrays probed with cdnas produced from hp versus total rna samples.
it should be noted that equal amounts of cdna are used to probe each microarray and the intensities are scaled so that each array has approximately the same average value.
this normalization will diminish the effect of reduced polysome abundance in the eif4g mutant versus wt cells.
the total amount of mrna could also decline in the mutant owing to reduced transcription or increased mrna turnover accompanying diminished translation, which would offset the effect of decreased polysome abundance on the calculated translational efficiencies.
hence, comparing te values can indicate absolute differences in translational efficiency between two genes in the same strain, but it reveals only relative differences in efficiency for a given gene between two strains.
as a quality control for the polysomal fractionation and mrna extraction procedures, we first analyzed the distribution of several mrnas among heavy polysomes, light polysomes , and 80s monosomes using real-time rt-pcr to quantify mrna concentrations .
the distributions of rpl41a and rpl41b mrnas were examined because their coding sequences, of only  <dig> nt  <cit> , are large enough to accommodate only two translating 80s ribosomes  <cit> , and at the average ribosome density for yeast mrnas they should generally contain only one translating 80s ribosome at a time  <cit> ; hence, the majority of these two mrnas should occur in the 80s monosome fraction.
the distributions of rpl41a and rpl41b mrnas observed for wt cells were highly similar to one another and displayed the expected preponderance of mrna in the 80s fractions and smaller proportions in the lp fractions .
by contrast, the hsp <dig>  pdc <dig>  and act <dig> mrnas were most abundant in the hp fractions and least abundant in the 80s or lp fractions, whereas hac <dig> mrna showed relatively equal abundance in all three fractions .
these findings are in accordance with previous polysomal profiling of these four mrnas  <cit> .
for microarray analysis, three biological replicates were examined , representing hp and total rna preparations from three independent pairs of wt and mutant cultures.
cy3-labeled cdnas were generated from the  <dig> hp and  <dig> total rna samples prepared for each strain and the resulting  <dig> sets of cdnas were used to probe three  replicate whole-genome microarrays, containing multiple 60-mer oligonucleotides for each gene .
the "normalized gene expression summary values" were calculated for each gene from the data obtained from the three technical replicates and used to calculate the translational efficiency  of each gene as the ratio of the intensity values for hp to total rna  for each project .
we first constructed ma plots  <cit>  to evaluate the reproducibility of mrna intensities measured for the biological replicates of each strain.
such plots display the ratios of mrna intensities between two arrays  as a function of the average intensities of the mrnas .
the variance  of m provides a measure of the range of intensity differences between two arrays across the genome.
representative ma plots are shown in figures 3a-b, and the variances are summarized in table s <dig> .
the comparisons of biological replicates from the same strain yielded relatively low σ <dig> values for both hp and total rna samples, that compare favorably with σ <dig> values reported previously for biological replicates of polysomal rna  <cit> .
we also used ma plots to compare the intensities of hp or total mrnas between mutant and wt cells, and the variances in these plots were substantially higher than the corresponding values for replicates from the same strain .
these latter plots indicate significant differences in the intensities of both total and hp mrnas between mutant and wt cells for a large fraction of the genome.
<dig> mean translational efficiencies were calculated by averaging te values from all three projects for each gene
finally, we constructed ma plots to quantify the differences in mrna abundance in polysomes versus total mrna, to visualize the variation in translational efficiency  across the genome for each strain.
interestingly, the σ <dig> values for the hp:t intensity ratios are ~2-fold higher for wt than for mutant cells , as illustrated in figure 3e-f. this was the first indication that the breadth of translational efficiencies  across the genome is reduced by depletion of eif4g.
to depict graphically the population of mrnas that are translated with relatively higher or lower efficiencies in wt versus mutant cells, we constructed scatter plots of hp/t ratios  for wt versus mutant mrnas using the mean te values calculated by averaging data from all three biological replicates .
the regression line of the scatter plot has a slope significantly larger than unity , which indicates that mrnas with greater than average te in wt   tend to be translated at relatively lower efficiencies in the mutant cells.
moreover, mrnas with lower than average te in wt  tend to be translated relatively better in the mutant.
considering the  <dig> genes with te values larger than the genome average in wild-type cells, the tewt/te4g ratio  is  <dig> .
for the remaining genes with te values smaller than the genome average, the mean tewt/te4g ratio is  <dig> .
as a consequence of these trends, there is a narrower range of translational efficiencies at both ends of the spectrum, in mutant versus wt cells.
this last conclusion was further supported by tabulating the numbers of mrnas with te values above or below unity between mutant and wt cells.
in wt,  <dig> mrnas have mean tes ≥  <dig> , and  <dig> mrnas have mean te values ≥  <dig>  .
in the mutant cells these gene categories are much smaller , indicating that a considerably smaller proportion of mrnas have higher than average translational efficiencies in the mutant cells.
a similar trend applies to mrnas with relatively low te values .
thus, the proportions of mrnas translated with either higher or lower than average translational efficiencies are reduced on depletion of eif4g.
the fact that the range of translational efficiencies is restricted by eif4g depletion implies that eif4g contributes to the higher than average te values for the most efficiently translated mrnas in wt cells.
to verify this deduction, we determined the proportion of the mrnas with tewt values ≥  <dig>  that are translated more efficiently in wt versus mutant cells, ie.
tewt ≥  <dig>  ∩ tewt > te4g.
this condition holds for > 97% of the  <dig> mrnas with tewt ≥  <dig> .
a similar conclusion emerged for the  <dig> mrnas with tewt ≤  <dig> , of which ~90% are translated less efficiently in wt than in mutant cells .
this last comparison confirms that the least efficiently translated group of mrnas in wt cells owe their relatively low te values, at least partly, to the presence of eif4g function.
below, we consider different mechanisms that could account for this negative effect of eif4g on translational efficiency.
only a small proportion of genes exhibit substantially altered translational efficiencies on depletion of eif4g
we focused next on the particular mrnas whose translational efficiencies differ the most between mutant and wt cells  because the difference in te between mutant and wt cells is modest for the majority of mrnas, coupled with the experimental variability in te values calculated from the different projects, there is a small fraction of genes for which the difference between mean te4g and tewt values calculated from all three projects is statistically significant.
we were able to identify  <dig> mrnas  that exhibit mean te4g/tewt ratios of ≤  <dig>  and for which the mean te4g value differed from the mean tewt value in all three projects with a p-value ≤  <dig>  in a two-tailed student's t-test, of which  <dig> mrnas differed with a p-value of ≤  <dig>  .
a subset of these  <dig> mrnas are listed in figure 5a , sorted on the mean te4g/tewt values .
note that most of these mrnas exhibit relatively high te values in wt cells  but display tes in the mutant closer to unity .
thus, these genes all exhibit higher than average translational efficiencies in wt cells that are reduced in the mutant to values closer to the genome-average te value .
we similarly identified  <dig> mrnas exhibiting a higher translational efficiency in the mutant versus wt, with mean te4g/tewt ratios ≥ <dig>  and for which the difference between the mean te4g and tewt values was significant at p ≤  <dig> , of which  <dig> differed with a p-value of ≤  <dig>  .
as illustrated in figure 5b, the majority of such mrnas exhibit lower than average translational efficiencies in wt cells with tewt values ≤  <dig>  , but efficiencies in the mutant that are closer to the genome-average te value .
thus, their relatively low te values in wt cells are increased on depletion of eif4g in the mutant.
these comparisons support the conclusion that eliminating eif4g narrows the range of translational efficiencies at both ends of the spectrum.
in an effort to validate the microarray measurements of te values, we conducted real-time qrt-pcr analysis of particular mrnas in the polysomal and total rna preparations used to produce the cy3-cdnas for probing microarrays.
we analyzed a set of  <dig> genes, most belonging to the two groups of genes just described with mean te4g values that are higher or lower than the cognate mean tewt values by a factor of  <dig>  or more.
as shown in figure s <dig> , the mrnas identified by microarray analysis with mean te4g/tewt ratios ≥ <dig>  displayed corresponding te4g/tewt ratios measured by qrt-pcr that were significantly greater than those for mrnas with mean te4g/tewt values of ≤  <dig>  in the microarray analysis.
thus, it appears that the microarray analysis reliably identified two groups of genes that are affected oppositely by depletion of eif4g.
characteristics of genes exhibiting altered translational efficiencies on depletion of eif4g
we wished next to determine whether the genes that displayed the largest differences in translational efficiencies between mutant and wt cells tend to be involved in common biological processes.
to this end, we conducted a gene ontology analysis using the mips funcat system , which determines whether genes of interest are significantly enriched in particular cellular functions.
analysis of the  <dig> genes with te4g/tewt ≥ <dig> , which are translated relatively better on eif4g depletion, revealed that they were enriched for genes with specific cellular functions  .
this encompasses genes involved in multiple key aspects of transcription and rna processing, such as the core transcriptional machinery , histone assembly or modification , transcription factors of the tor growth control pathway , and components of the tho mrna export complex , as well as dna processing components, especially as involved in control of dna topology .
similarly enriched were genes involved in plasma membrane related trafficking, both endocytosis  and exocytosis .
many of these processes correspond to key housekeeping functions, explaining the enrichment for essential genes  evident in figure 6b.
whether the increased translational efficiency of these housekeeping genes following depletion of eif4g is a consequence of relief from translational repression exerted by eif4g, or if it corresponds to a more general cellular effort to counter the effects of loss of eif4g, is not clear.
notably, the  <dig> genes translated less efficiently following depletion of eif4g tended not to encompass essential genes , and several housekeeping processes, such as dna processing and protein modification were underrepresented in this group .
in contrast, it was enriched for genes involved in oxidative stress response, especially components of the cellular peroxidase/thioredoxin systems, such as gpx <dig>  hyr <dig>  trx <dig>  srx <dig> and tsa <dig>  these findings suggest that under conditions of eif4g down-regulation, a select group of mrnas whose products function in housekeeping processes such as transcription and dna processing, are translated relatively better than all other mrnas; whereas a group of non-essential genes involved in cellular energy production are translated relatively worse.
given the reported involvement of eif4g in activating mrnas for recruitment of the 43s pic and scanning the 5'utr, we examined the two sets of genes with significantly altered te4g/tewt ratios to determine whether they exhibit atypical 5'utr lengths or secondary structures.
we employed the database of 5'utr lengths for  <dig> yeast orfs from lawless et al  compiled from results of genome-wide studies of 5' transcription start sites.
interestingly, for the  <dig> genes with te4g/tewt ≥ <dig>  whose features were compiled by lawless et al, the mean 5' utr length is  <dig> ±  <dig> nt, which is ~ <dig>  fold greater than the average 5'utr length of  <dig> ±  <dig>  nt for all  <dig> genes in the database   <cit> .
for the  <dig> genes with te4g/tewt values ≤  <dig> , the mean 5' utr length is  <dig> ±  <dig> nt, significantly smaller than that determined for the genes with te4g/tewt ≥ <dig>   but not significantly different than the mean value for all mrnas.
the enrichment for long 5'utr lengths for genes with te4g/tewt ≥ <dig>  is evident in figure  <dig>  where their length distribution is compared to that of all  <dig> 5'utrs .
thus, the fraction of genes exhibiting a relative increase in te in the mutant have a significantly longer than average 5'utr, whereas those exhibiting a relative decrease in te on eif4g depletion have a nearly typical length distribution.
thus, the class of mrnas most dependent on eif4g exhibit the comparatively short 5'utr lengths characteristic of the majority of yeast mrnas.
using the computer program randfold to predict folding of 5'utrs, lawless et al reported that the vast majority of yeast 5' utrs appear not to be strongly folded, with only  <dig> 5'utrs showing low minimum free energies  of folding with an associated p-value of <  <dig> .
none of these mrnas was found among the genes showing the greatest reductions in te in the mutant versus wt .
in fact, four of these  <dig> mrnas, all containing long 5'utrs  with strong predicted secondary structures, appear to be translated more efficiently on depletion of eif4g, showing mean te4g/tewt ratios of  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  , and  <dig>  ±  <dig>  .
these results do not support the possibility that the translation of mrnas with highly stable secondary structures in their 5'utrs would be strongly enhanced by eif4g.
it has been reported that mammalian eif4g plays a critical role in the ability of post-termination 40s subunits to resume scanning following translation of a short uorf  <cit> .
hence, we asked whether the genes whose translation is relatively lower in the mutant versus wt might display an atypical occurrence of uorfs.
for the  <dig> genes with te4g/tewt values ≤  <dig>  whose occurrences of uorfs were tabulated by lawless et al  <cit> , there is an average of  <dig>  ±  <dig>  uorfs per transcript.
for the  <dig> genes with te4g/tewt ≥ <dig> , the corresponding average is  <dig>  ±  <dig>  uorfs per transcript.
neither of these frequencies differs significantly from the average uorf occurrence of  <dig>  ±  <dig>  uorfs per transcript tabulated for  <dig> genes by lawless et al.
thus, we found no indication that the presence or absence of uorfs is a critical determinant of the effect of eif4g on the translational efficiency of eif4g-responsive mrnas.
in animals, translational control of specific mrnas frequently involves trans-acting factors that bind to specific recognition elements in the 3'utr and target eif4f assembly at the cap structure  <cit> .
accordingly, we examined whether the 3'utr length differs significantly between the two sets of genes identified above.
as shown in figure  <dig>  the 3'utr length appears to be slightly smaller for the group of genes with te4g/tewt ≤  <dig>  versus that with te4g/tewt ≥ <dig>  ; however, neither group displays a mean 3'utr length that is significantly different from that of all genes.
hence, it seems unlikely that 3'utr length is an important parameter in determining the dependence of translational efficiency on eif4g.
finally, we examined  <dig> mrnas reported to have an a-rich ires  and also the ires-containing mrna ure <dig>  to determine whether the translational efficiencies of these mrnas might be increased or decreased on depletion of eif4g.
we observed no significant deviation from unity in the te4g/tewt ratios of the  <dig> genes with a-rich iress:  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  ,  <dig>  ±  <dig>  .
the same holds true for the ure2/ynl229c mrna, exhibiting a te4g/tewt ratio of  <dig>  +  <dig>  .
these findings provide no indication that eif4g is a critical factor for the function of these ires elements, although it is unknown whether any of the iress are functional under the nonpermissive growth conditions of our experiments.
most genes with short orfs require eif4g to achieve their characteristic, higher than average translational efficiencies
because we examined polysomal rnas present in "heavy polysomes" , genes whose transcripts contain, on average, less then  <dig> translating ribosomes were likely underrepresented in this analysis.
of particular concern are those genes with relatively long coding regions whose mrnas have an average of  <dig> or fewer translating ribosomes in wt cells, i.e., with low ribosome densities, because such inefficiently translated mrnas might be particularly dependent on eif4g.
to address this possibility, we extended our microarray analysis to include rna isolated from the "light polysome"  fractions obtained from the same gradients that yielded the hp fractions analyzed above .
the mean te for each gene was calculated as the ratio of lp/t rna intensities in all three projects, as above for hp rna.
we then cross-referenced the resulting te values with a database listing the ribosome densities of  <dig>  yeast genes described by arava et al.
<cit> , focusing on a group of  <dig> genes whose mrnas in that study displayed peak occupancies of only 1- <dig> ribosomes per mrna, and thus should occur primarily in the lp fractions of our study, of which  <dig> were interrogated on our microarrays.
a subset of  <dig> genes from this group contain relatively long coding sequences  and exhibit average ribosome densities of ≤  <dig>  ribosomes per  <dig> nt-well below the genome-average density of  <dig>   <cit> .
the mean tewt calculated for these genes from our lp data,  <dig>  +  <dig> , is significantly below the genome average tewt value of  <dig>  ±  <dig>  derived from the hp data for all  <dig> orfs, indicating that these genes exhibit an atypically low proportion of mrna associated with ribosomes in addition to a low ribosome density.
consistent with our findings on mrnas in hp fractions, the majority of these poorly translated mrnas in the lp fractions exhibit higher te values in the eif4g mutant versus wt cells .
thus, it appears that eif4g is not a critical rate-limiting factor for this group of very inefficient mrnas.
we also examined a subset of  <dig> genes from the group of  <dig> mentioned above, which exhibit peak occupancies of only 1- <dig> ribosomes per mrna simply because they have short orf lengths , as their mean ribosome density  actually exceeds the genome-average of  <dig>   <cit> .
interestingly, these genes have a mean tewt value of  <dig>  ±  <dig>  , that is substantially higher than the genome-average tewt value  and most of these genes have significantly lower te values in the eif4g mutant versus wt  .
having identified a group of efficiently translated mrnas with a marked dependence on eif4g that contain atypically short coding sequences, we examined the behavior of all genes with short orfs  in both the lp and hp data sets.
as illustrated in the log-log plots of figure s <dig> , ~90% of these genes exhibit te values greater than unity in wt cells , compared to only 55% for genes of all orf lengths .
this disparity reflects the broader phenomenon that tewt values are inversely related to orf length, as revealed in the scatterplot of tewt values versus orf length for the entire hp dataset .
this relationship is not unexpected, as it was noted previously that ribosome densities on mrnas  <cit>  and protein expression levels  <cit>  are inversely related to orf length in yeast.
interestingly, the majority of the short-orf genes exhibit reductions in te values in the eif4g mutant of ~10% on average , similar to the average reduction in te mentioned above for all genes with tewt values above unity .
the reduction in te evoked by depletion of eif4g for small-orf genes is also obvious in the scatterplots of figure s <dig> , as dampening te values for the shortest orf lengths in the eif4g mutant is observed.
thus, genes with short orfs tend to be translated more efficiently in wt cells and to be dependent on eif4g for their maximum efficiency.
it is noteworthy that the two sets of ~ <dig> genes we identified above displaying the greatest changes in te values on depletion of eif4g differ dramatically in average orf length.
the group exhibiting the greatest reductions in translation efficiency  has a mean orf length below the genome average by nearly a factor of two , while genes showing the greatest increases in efficiency  have a mean orf length 70% larger than average  .
these findings suggest that orf length, in addition to 5'utr length, determines the influence of eif4g on translational efficiency.
below, we propose a molecular explanation for this finding, based on the known relationship between transcript length and the stability of eif4f-cap interaction  <cit> .
considering the strong correlation between orf length and effect of eif4g depletion on translational efficiency shown in figure  <dig>  it seems possible that the enrichment of cellular functions associated with the gene sets exhibiting te4g/tewt ≤  <dig>  or te4g/tewt ≥ <dig>  described above  could at least partially reflect a preponderance of genes with unusually small or large orf lengths in those functional categories.
discussion
in this study, we have examined the genome-wide consequences for translational efficiency of simultaneously eliminating eif4g <dig> and depleting eif4g <dig> from yeast cells.
the conditional depletion of eif4g <dig> achieved using a degron-tagged version of this protein was highly effective and reduced the polysome content and rate of translation to only 20-30% of wt levels, indicating a substantial reduction in the rate of translation initiation.
we used genome expression microarrays to measure the abundance of each mrna in heavy polysomes  relative to its level in total mrna to calculate translational efficiencies of  <dig> different genes.
the results indicated that the overwhelming majority of mrnas experienced only a moderate change in translational efficiency on eif4g depletion.
less than 2% of the genes showed a statistically significant decrease in te in the mutant by a factor of  <dig>  of more, and the genes in this group that were affected the most displayed reductions of a factor of ~ <dig>  or less.
while the actual percentage of genes affected to this extent is probably higher, only ~10% of genes exhibited decreases in te of this magnitude for each biological replicate, which likely represents the upper size limit for this category.
thus, we did not detect even a small group of mrnas that are dramatically dependent on eif4g for translation in vivo.
we made the unexpected observation, however, that depletion of eif4g narrows the range of translational efficiencies for a large fraction of mrnas, decreasing the number with efficiencies that are substantially higher or lower than the genome-average te.
this trend is well illustrated in the log-log plots of mean te values in wt versus mutant cells , and also by the fact that depleting eif4g reduced  the numbers of mrnas with te values either  <dig> -fold higher, or  <dig> -fold lower, than unity .
furthermore, the bulk of mrnas with te values ≥  <dig>  in wt cells are, at least to some extent, dependent on eif4g for their higher than average te values .
this dependence is consistent with a significant role for eif4g in stimulating one or more steps of initiation for the most efficiently translated mrnas in the cell, presumably the activation of mrna for recruitment of the 43s pic, scanning the 5'utr, or start codon recognition.
unexpectedly, we found that many mrnas with lower than average te values in wt cells exhibit an increased translational efficiency on eif4g depletion.
it is conceivable that eif4g directly impairs the translation of these latter mrnas.
however, we favor an indirect mechanism involving competition among all mrnas for limiting initiation factors or pics, coupled with the role of eif4g in stimulating efficiently translated mrnas at the expense of those with lower than average efficiencies.
in the absence of eif4g, this competitive edge would be eliminated for the first group and thereby enable the second group of mrnas to compete better for limiting factors/pics.
the small group of ~ <dig> genes we identified that are most dependent on eif4g for their higher than average tes in wt cells contain a mean 5'utr length that is slightly below the genome-average for all mrnas , a feature that should facilitate efficient scanning and aug recognition.
this was surprising because we expected to find that the mrnas most dependent on eif4g would have long or highly structured 5'utrs, requiring the eif4g·eif4a complex for unwinding secondary structure to promote 43s attachment or scanning.
in fact, the ~ <dig> genes we identified whose translation is stimulated the most by eliminating eif4g contain a mean 5'utr length substantially larger than the genome average .
the fact that these latter mrnas display a lower than average te in wt cells and benefit from the absence of eif4g seems to indicate that they function inefficiently at steps of initiation not significantly enhanced by eif4g.
given their long 5' utr lengths, it seems likely that scanning to the start codon is relatively inefficient for these mrnas.
if so, then the fact that depleting eif4g does not exacerbate this deficiency suggests that factors besides eif4g are more critically required for efficient scanning through long 5'utrs in yeast.
this last suggestion is consistent with our finding that none of the  <dig> mrnas predicted by the randfold program to contain the most stable secondary structures among yeast 5' utrs  <cit>  displayed a significant reduction in te on eif4g depletion--in fact, four such mrnas appear to be translated more efficiently on eif4g depletion.
thus, other initiation factors besides eif4g might also be more critically involved in removing secondary structures in advance of the scanning pic.
this view is supported by the fact that in a mammalian reconstituted system, eif4g, eif4a and eif4b are sufficient for 43s attachment and scanning on β-globin mrna, which harbors a relatively unstructured 5'utr, whereas the dexh-box protein dhx <dig> is required for initiation complex assembly on mrnas containing more structured 5'utrs  <cit> .
similarly, there is evidence that yeast dead-box protein ded <dig> contributes more than eif4a does to the processivity of scanning in vivo  <cit> .
these findings are in agreement with the possibility that the eif4e/eif4g/eif4a complex  is more critical for 43s pic attachment near the 5' end of the mrna than for subsequent scanning to the start codon.
thus, our results are consistent with the model that 43s attachment is a rate-limiting step for a large proportion of mrnas with higher than average tes, and that this step is stimulated by eif4g, particularly for the ~ <dig> genes we identified with the greatest dependence on eif4g that contain relatively short 5'utrs.
by contrast, scanning or aug recognition would be rate-limiting for mrnas with longer than average 5'utrs whose translation is enhanced by depletion of eif4g, because these steps are not critically dependent on eif4g.
the fact that eliminating eif4g mitigates the lower than average translational efficiencies of this second group of mrnas can be explained by proposing that the negative effect of depleting eif4g on 43s attachment is outweighed by their enhanced ability to compete with other mrnas for limiting factors that promote scanning or aug recognition.
fulfilling this last stipulation of our model would be facilitated if the inefficient mrnas with long 5'utrs are relatively ineffective at exploiting eif4g function in 43s attachment.
that is, if eif4g contributes relatively less to 43s attachment by these inefficient mrnas in wt cells, then depleting eif4g would produce relatively smaller reductions in their translation rate.
one reason for thinking that this condition holds is our finding that this group of mrnas also displays unusually long coding sequences, whereas the mrnas we identified with the greatest dependence on eif4g exhibit smaller than average orf lengths.
recent findings by jacobson et al  <cit>  indicate that shorter yeast mrnas produce more stable eif4f-cap interactions than do longer mrnas, which is fully dependent on an extended poly tail and pabp.
presumably, shorter mrnas more efficiently assemble a closed-loop mrnp via pabp-eif4g interaction, which stabilizes eif4f binding to mrna  <cit> .
in fact, the possibility of less efficient 5'-3' interaction for larger mrnas was advanced previously as one explanation for the inverse correlation between ribosome density and orf length  <cit> , which we confirmed here using te values .
hence, we suggest that longer mrnas are affected less than shorter mrnas by the elimination of eif4g because the eif4f-cap interaction is inherently less stable for longer transcripts and, hence, less efficacious in promoting 43s recruitment when eif4g is present.
the fact that depleting eif4g diminishes, but does not eliminate the correlation between te and orf length  indicates that reduced eif4g-pabp interaction is not the only factor limiting the translation of mrnas with longer orfs, and limited processivity of elongating ribosomes or less efficient termination have been suggested as other possibilities  <cit> .
we showed previously that depletion of eif4g did not lower the amounts of native 48s complexes containing the rpl41a or mfa <dig> mrnas  <cit> , both very short transcripts, which is ostensibly at odds with the idea that eif4g has an important function in 43s attachment to mrna.
examining the results we obtained for these mrnas in the lp dataset  reveals that they both exhibit mean te4g values ~90% of their tewt values .
thus, even if we assume that these two mrnas require eif4g only at the step of 43s attachment to achieve their maximum translation rates, it would have been very difficult to detect a 10% decrease in the levels of their free 48s complexes with the techniques employed in the previous study  <cit> .
it remains to be determined what features in mrna, besides a short 5'utr and short orf length, are responsible for the more pronounced requirement for eif4g displayed by the small fraction of yeast mrnas identified here.
considering that eif4g is essential in yeast, and also noting its role as a protein bridge linking the eif4e-mrna-pabp mrnp to components of the 43s complex   <cit> , it is surprising that a significant amount of translation still proceeds in the absence of this factor.
based on our microarray data, it appears that eif4g is dispensable for the translation of most, if not all mrnas in vivo, indicating that it is rate-enhancing rather than essential in budding yeast.
this stands in contrast to the critical requirement for the eif <dig> complex, which is required for nearly all translation in yeast, and is crucial for attachment of native 43s complexes to mrnas  that can assemble 48s pics in cells depleted of eif4g  <cit> .
of course, we cannot exclude the possibility that a compensatory initiation pathway comes into play during the  <dig> h of incubation in the non-permissive conditions used to thoroughly deplete eif4g.
it is also impossible to eliminate the possibility that a very small fraction of the wt amount of eif4g, below the detection limit of our western analysis, is sufficient to catalyze the residual protein synthesis that occurs in the depleted cells.
this seems unlikely, however, because the eif4g level in wt cells is already lower than those of nearly all other initiation factors  <cit> .
on the other hand, the  <dig> to 4-fold reduction in the rate of translation, and the narrowed range of translational efficiencies evoked by depletion of eif4g, could have serious consequences for a subset of dosage-sensitive proteins with essential functions in the cell.
moreover, cell division could be blocked under these conditions by regulatory mechanisms that respond to a drop in the rate of synthesis of a key cell cycle controlling factor, eg., the g <dig> cyclin cln <dig>  <cit> .
considering that cell division is not blocked by a decrease in the overall translation rate of ~70% occurring in response to hyperosmotic stress  <cit> , eif4g depletion might evoke a comparatively greater reduction in translation of a key protein required for cell division than occurs during osmotic stress.
given that depletion of eif4g reduces the translation rate by  <dig> to 4-fold, it is surprising that the average te calculated for all  <dig> genes decreased only a small amount, from  <dig>  ±  <dig>  in wt cells to  <dig>  ±  <dig>  in the mutant.
of course, many genes translated with higher than average efficiencies in wt exhibit much larger reductions in te values on depletion of eif4g, but this effect was counterbalanced by increased translation of many genes with lower than average tewt values.
as noted above, the fact that microarray results are normalized to give each array the same average signal intensity will dampen the reduction in polysomal mrna abundance in the eif4g mutant, and the amounts of total mrna might also decline on eif4g depletion, which would offset the effect of decreased polysomal mrna on the calculated te values.
it is also conceivable that eif4g depletion triggers a signal transduction response that decreases the rate of elongation, counteracting the effect of reduced initiation on polysome size.
for example, oxidative stress reduces the rates of both initiation and elongation in yeast  <cit> .
because we examined cells lacking eif4g <dig> and depleted of eif4g <dig>  it could be argued that the changes in translational efficiencies we observed result primarily from the absence of only eif4g <dig> or eif4g <dig> rather than the elimination of both eif4g isoforms.
this is unlikely in view of recent findings by clarkson et al on mutant strains expressing only eif4g <dig> or eif4g <dig> and engineered to express each isoform at a level equivalent to the combination of both isoforms in wt.
these strains displayed almost no changes in translational efficiency genome-wide  <cit> , providing strong evidence against the possibility that eif4g <dig> or eif4g <dig> is specifically required to support the translation of particular mrnas.
in this same study, two groups of protein-coding genes  displayed a significant change in translational efficiency on deletion of only tif <dig>  encoding the major isoform , which reduced the growth rate and polysome content relative to the isogenic wt strain.
only 10% of the genes with significantly repressed translational efficiencies in tif4631Δ cells thus identified by clarkson et al belong to the group of ~ <dig> genes we identified here with mean te4g/tewt ratios of ≤  <dig> .
however, the group of translationally repressed genes in the clarkson et al study displayed an average te4g/tewt ratio in our experiments  that is significantly below the genome-average te4g/tewt ratio  and also the average te4g/tewt ratio determined in our experiments for the group of translationally enhanced genes identified by clarkson et al .
thus, the translational efficiencies of at least a subset of genes are affected similarly by the absence of eif4g <dig> alone and the elimination of both eif4g <dig> and eif4g <dig> simultaneously.
this is consistent with the conclusion that eif4g <dig> and eif4g <dig> perform essentially identical functions  <cit> .
a recent analysis of the consequences of depleting eif4gi and eif4gii with sirnas in cultured mammalian cells  <cit>  reached certain conclusions congruent, and others that seem to differ, from our findings.
it was found that depleting both eif4gi and eif4gii reduced overall translation by only ~20%, whereas depleting two eif <dig> subunits provoked a stronger  reduction, consistent with the greater requirement for eif <dig> versus eif4g we observed in yeast  <cit> .
eif4gi depletion reduced the translational efficiencies of a subset of mammalian mrnas, including a group whose products function in mitochondrial regulation, bioenergetics, and cell proliferation.
in accordance with our observations, there was no significant correlation between the presence of long or structured 5'utrs and the degree of eif4gi-dependence.
this is consistent with the aforementioned suggestion that eif4gi is more important for 43s attachment than for subsequent scanning through the 5'utr.
at odds with our results, however, the eif4gi-dependent class of mrnas appeared to be somewhat enriched in those containing uorfs, and the presence of an uorf was shown to increase the eif4gi dependence on translation.
one possibility is that the majority of uorf-containing mrnas in yeast do not support appreciable reinitiation in wt cells, as this process has strict requirements for uorf length and cis-acting sequences surrounding the stop codon  <cit> .
in this event, eliminating the potential role of eif4g in stimulating reinitiation would be difficult to detect on a genome-wide basis in yeast.
CONCLUSIONS
our results indicate that eliminating both isoforms of eif4g from yeast cells elicits a substantial reduction in the rate of translation initiation that is severe enough to block cell division, but does not evoke dramatic changes in the relative translational efficiencies of the majority of mrnas.
rather, we observed a large-scale narrowing of translational efficiencies, including mrnas with higher or lower than average efficiencies, which is expected to disturb the stoichiometry of protein components comprising many cellular pathways and structures.
our finding that mrnas with the greatest dependence on eif4g are relatively well-translated, do not contain long or highly structured 5'utr, and also have short coding sequences, is consistent with the idea that eif4f is most critically required to enhance 43s attachment to the mrna 5' end rather than for scanning through long, structured 5' utrs.

 translation of most mrnas in eukaryotic cells occurs by a scanning mechanism wherein the small ribosomal subunit recruits methionyl initiator trna in a ternary complex with eif2-gtp, in a reaction stimulated by other eifs, and the resulting 43s pre-initiation complex binds near the m g-cap structure of the mrna to assemble the 48s pic. attachment of the 43s complex at the mrna 5' end is stimulated by the eif4f complex, comprised of cap-binding protein eif4e, the scaffold subunit eif4g, and the dexd/h-box helicase eif4a, which is thought to provide a single-stranded region in the mrna for recruiting the ribosome. binding sites in eif4g for either eif or eif and eif are thought to facilitate recruitment of the 43s pic to eif4f bound at the cap structure. eif4g also harbors a binding site for the poly-binding protein that, together with an rna binding domain in the middle region of mammalian eif4g, increases the stability of eif4f binding to the mrna 5' end and also mediates circularization of mrna in the activated eif4f·mrna·pabp mrnp. in addition to stimulating recruitment of the 43s pic to the mrna 5' end, there is evidence that the atp-dependent rna helicase activity of eif4a facilitates ribosomal scanning through secondary structures in the 5' utr to enhance recognition of the aug start codon. however, other dexd/h helicases have been implicated in scanning through long or structured 5' utrs, including ded1/ddx in yeast and dhx in mammals, and it is uncertain whether eif4a and its binding partners in eif4f are critically required for scanning. in fact, 43s recruitment and location of the start codon has been reconstituted in vitro for an artificial mrna with an unstructured 5' utr in the absence of eif4f, eif4a, eif4b, and atp, requiring only the eif2·gtp·met-trnai met ternary complex, eif eif and eif1a. hence, it is possible that native mrnas devoid of stable structures in the 5'utr could be translated at relatively high efficiencies in the absence of eif4f. indeed, we showed previously that genetically depleting eif4g from yeast cells reduces general translation initiation but does not impair 48s pic formation in vivo by two native mrnas. based on its presumed functions in mrna activation and scanning, it is generally assumed that eif4f plays an important role in determining the relative efficiencies of translation among the repertoire of cellular mrnas and, hence, is a key factor in translational control of gene expression. we examined this hypothesis in yeast by measuring the effect of genetically depleting eif4g from yeast cells on translational efficiencies of mrnas genome-wide. the depletion of eif4g was very effective and it reduced protein synthesis rates by a factor of ~ leading to cell growth arrest. surprisingly, however, the translational efficiencies of most mrnas were not substantially affected by eif4g depletion. an intriguing consequence of a strong reduction in eif4g levels was to narrow the range of translational efficiencies genome-wide by reducing the translation of many mrnas with higher than average translational efficiencies in wild-type cells while increasing the translation of different mrnas that are normally translated with lower than average efficiencies. our findings suggest that eif4g is not essential for translation of any mrnas in yeast cells, but it enhances the differentiation of translational efficiencies among cellular mrnas.  depletion of eif4g in cells lacking eif4g evokes a marked decrease in the rate of translation initiation in vivo to examine the consequences for global translation of eliminating both isoforms of eif4g, we employed a strain deleted of the chromosomal gene encoding eif4g and harboring a temperature-sensitive degron allele of the gene encoding eif4g. the tif4631-td allele encodes ubiquitin and a thermolabile dihydrofolate reductase moiety fused to the n terminus of eif4g expressed from a copper-dependent promoter, and is integrated into the chromosome in a manner that disrupts the resident wild-type tif allele. the strain also contains a galactose-inducible form of the gene encoding the ubiquitin ligase required for proteasomal degradation of degron-tagged proteins by the "n-end rule" pathway. shifting cells from medium containing copper and raffinose at 25°c to medium containing galactose and raffinose but lacking copper at 36°c represses new synthesis and triggers proteasomal degradation of the existing degron-tagged eif4g1-td protein. we showed previously that under non-permissive conditions this degron mutant cannot form colonies from single cells, exhibits a strong reduction in doubling time within h, and essentially ceases growth and division by h after the shift to non-permissive conditions. this growth arrest can be reversed by shifting cells back to permissive conditions. consistent with our previous results, incubation for h under non-permissive conditions was required to deplete eif4g1-td in whole cell extracts below the detection limit of western analysis. note that both the wild-type and mutant wces appear to contain an n-terminally truncated form of eif4g that migrates more rapidly than either the wt or degron-tagged full-length proteins. because this truncation is subject to degradation in the degron mutant, but necessarily lacks the n-terminal modifications necessary for n-end rule degradation, it is likely generated from the full-length proteins in vitro following cell lysis. after h of depletion, the degron mutant exhibits the expected reduction in total polysomes and commensurate increase in 80s monosomes, leading to a decreased ratio of polysomes to monosomes by a factor > compared to the p/m ratio for the wt strain under the same conditions. this is the stereotypical consequence of selective impairment of translation initiation, involving a decrease in new initiation events, "run-off" of elongating ribosomes from existing polysomes, and subsequent accumulation of excess free subunits as 80s couples. note that depletion of two essential subunits of the eif complex, in a separate mutant expressing degron-tagged forms of these proteins, evokes a more complete polysome run-off than observed in the eif4g1-td mutant. consistent with the polysome profiles, the rate of total protein synthesis, measured by incorporation of radioactive methionine into acid-insoluble material, was reduced in the eif4g1-td mutant to ~30% of the wt value after h in the non-permissive condition, whereas the eif degron mutant displayed no detectable -met incorporation under these conditions. thus, in accordance with our previous conclusions, depletion of eif4g in cells lacking eif4g leads to a marked reduction in the rate of translation initiation, but one less severe than that provoked by a comparable depletion of eif subunits. depletion of eif4g narrows the range of mrna translational efficiencies genome-wide although a significant level of translation continues following the extensive depletion of eif4g in the degron mutant, it was possible that translation of some mrnas would be greatly diminished while translation of others would continue relatively unaffected or even increase. to address this possibility, we determined the effect of depleting eif4g on the translational efficiencies of mrnas genome-wide. to this end, we conducted microarray analysis on rna isolated from the "heaviest" polysomes, containing or more elongating 80s ribosomes per mrna, and also total rna from wces, from both degron mutant and wt cells cultured for h under non-permissive conditions. translational efficiencies were calculated for each gene as the ratio of hybridization intensities on microarrays probed with cdnas produced from hp versus total rna samples. it should be noted that equal amounts of cdna are used to probe each microarray and the intensities are scaled so that each array has approximately the same average value. this normalization will diminish the effect of reduced polysome abundance in the eif4g mutant versus wt cells. the total amount of mrna could also decline in the mutant owing to reduced transcription or increased mrna turnover accompanying diminished translation, which would offset the effect of decreased polysome abundance on the calculated translational efficiencies. hence, comparing te values can indicate absolute differences in translational efficiency between two genes in the same strain, but it reveals only relative differences in efficiency for a given gene between two strains. as a quality control for the polysomal fractionation and mrna extraction procedures, we first analyzed the distribution of several mrnas among heavy polysomes, light polysomes, and 80s monosomes using real-time rt-pcr to quantify mrna concentrations. the distributions of rpl41a and rpl41b mrnas were examined because their coding sequences, of only nt, are large enough to accommodate only two translating 80s ribosomes, and at the average ribosome density for yeast mrnas they should generally contain only one translating 80s ribosome at a time; hence, the majority of these two mrnas should occur in the 80s monosome fraction. the distributions of rpl41a and rpl41b mrnas observed for wt cells were highly similar to one another and displayed the expected preponderance of mrna in the 80s fractions and smaller proportions in the lp fractions. by contrast, the hsp pdc and act mrnas were most abundant in the hp fractions and least abundant in the 80s or lp fractions, whereas hac mrna showed relatively equal abundance in all three fractions. these findings are in accordance with previous polysomal profiling of these four mrnas. for microarray analysis, three biological replicates were examined, representing hp and total rna preparations from three independent pairs of wt and mutant cultures. cy3-labeled cdnas were generated from the hp and total rna samples prepared for each strain and the resulting sets of cdnas were used to probe three replicate whole-genome microarrays, containing multiple 60-mer oligonucleotides for each gene. the "normalized gene expression summary values" were calculated for each gene from the data obtained from the three technical replicates and used to calculate the translational efficiency of each gene as the ratio of the intensity values for hp to total rna for each project. we first constructed ma plots to evaluate the reproducibility of mrna intensities measured for the biological replicates of each strain. such plots display the ratios of mrna intensities between two arrays as a function of the average intensities of the mrnas. the variance of m provides a measure of the range of intensity differences between two arrays across the genome. representative ma plots are shown in figures 3a-b, and the variances are summarized in table s. the comparisons of biological replicates from the same strain yielded relatively low σ values for both hp and total rna samples, that compare favorably with σ values reported previously for biological replicates of polysomal rna. we also used ma plots to compare the intensities of hp or total mrnas between mutant and wt cells, and the variances in these plots were substantially higher than the corresponding values for replicates from the same strain. these latter plots indicate significant differences in the intensities of both total and hp mrnas between mutant and wt cells for a large fraction of the genome. mean translational efficiencies were calculated by averaging te values from all three projects for each gene finally, we constructed ma plots to quantify the differences in mrna abundance in polysomes versus total mrna, to visualize the variation in translational efficiency across the genome for each strain. interestingly, the σ values for the hp:t intensity ratios are ~2-fold higher for wt than for mutant cells, as illustrated in figure 3e-f. this was the first indication that the breadth of translational efficiencies across the genome is reduced by depletion of eif4g. to depict graphically the population of mrnas that are translated with relatively higher or lower efficiencies in wt versus mutant cells, we constructed scatter plots of hp/t ratios for wt versus mutant mrnas using the mean te values calculated by averaging data from all three biological replicates. the regression line of the scatter plot has a slope significantly larger than unity, which indicates that mrnas with greater than average te in wt tend to be translated at relatively lower efficiencies in the mutant cells. moreover, mrnas with lower than average te in wt tend to be translated relatively better in the mutant. considering the genes with te values larger than the genome average in wild-type cells, the tewt/te4g ratio is. for the remaining genes with te values smaller than the genome average, the mean tewt/te4g ratio is. as a consequence of these trends, there is a narrower range of translational efficiencies at both ends of the spectrum, in mutant versus wt cells. this last conclusion was further supported by tabulating the numbers of mrnas with te values above or below unity between mutant and wt cells. in wt, mrnas have mean tes ≥, and mrnas have mean te values ≥. in the mutant cells these gene categories are much smaller, indicating that a considerably smaller proportion of mrnas have higher than average translational efficiencies in the mutant cells. a similar trend applies to mrnas with relatively low te values. thus, the proportions of mrnas translated with either higher or lower than average translational efficiencies are reduced on depletion of eif4g. the fact that the range of translational efficiencies is restricted by eif4g depletion implies that eif4g contributes to the higher than average te values for the most efficiently translated mrnas in wt cells. to verify this deduction, we determined the proportion of the mrnas with tewt values ≥ that are translated more efficiently in wt versus mutant cells, ie. tewt ≥ ∩ tewt > te4g. this condition holds for > 97% of the mrnas with tewt ≥. a similar conclusion emerged for the mrnas with tewt ≤, of which ~90% are translated less efficiently in wt than in mutant cells. this last comparison confirms that the least efficiently translated group of mrnas in wt cells owe their relatively low te values, at least partly, to the presence of eif4g function. below, we consider different mechanisms that could account for this negative effect of eif4g on translational efficiency. only a small proportion of genes exhibit substantially altered translational efficiencies on depletion of eif4g we focused next on the particular mrnas whose translational efficiencies differ the most between mutant and wt cells because the difference in te between mutant and wt cells is modest for the majority of mrnas, coupled with the experimental variability in te values calculated from the different projects, there is a small fraction of genes for which the difference between mean te4g and tewt values calculated from all three projects is statistically significant. we were able to identify mrnas that exhibit mean te4g/tewt ratios of ≤ and for which the mean te4g value differed from the mean tewt value in all three projects with a p-value ≤ in a two-tailed student's t-test, of which mrnas differed with a p-value of ≤. a subset of these mrnas are listed in figure 5a, sorted on the mean te4g/tewt values. note that most of these mrnas exhibit relatively high te values in wt cells but display tes in the mutant closer to unity. thus, these genes all exhibit higher than average translational efficiencies in wt cells that are reduced in the mutant to values closer to the genome-average te value. we similarly identified mrnas exhibiting a higher translational efficiency in the mutant versus wt, with mean te4g/tewt ratios ≥ and for which the difference between the mean te4g and tewt values was significant at p ≤, of which differed with a p-value of ≤. as illustrated in figure 5b, the majority of such mrnas exhibit lower than average translational efficiencies in wt cells with tewt values ≤, but efficiencies in the mutant that are closer to the genome-average te value. thus, their relatively low te values in wt cells are increased on depletion of eif4g in the mutant. these comparisons support the conclusion that eliminating eif4g narrows the range of translational efficiencies at both ends of the spectrum. in an effort to validate the microarray measurements of te values, we conducted real-time qrt-pcr analysis of particular mrnas in the polysomal and total rna preparations used to produce the cy3-cdnas for probing microarrays. we analyzed a set of genes, most belonging to the two groups of genes just described with mean te4g values that are higher or lower than the cognate mean tewt values by a factor of or more. as shown in figure s, the mrnas identified by microarray analysis with mean te4g/tewt ratios ≥ displayed corresponding te4g/tewt ratios measured by qrt-pcr that were significantly greater than those for mrnas with mean te4g/tewt values of ≤ in the microarray analysis. thus, it appears that the microarray analysis reliably identified two groups of genes that are affected oppositely by depletion of eif4g. characteristics of genes exhibiting altered translational efficiencies on depletion of eif4g we wished next to determine whether the genes that displayed the largest differences in translational efficiencies between mutant and wt cells tend to be involved in common biological processes. to this end, we conducted a gene ontology analysis using the mips funcat system, which determines whether genes of interest are significantly enriched in particular cellular functions. analysis of the genes with te4g/tewt ≥, which are translated relatively better on eif4g depletion, revealed that they were enriched for genes with specific cellular functions. this encompasses genes involved in multiple key aspects of transcription and rna processing, such as the core transcriptional machinery, histone assembly or modification, transcription factors of the tor growth control pathway, and components of the tho mrna export complex, as well as dna processing components, especially as involved in control of dna topology. similarly enriched were genes involved in plasma membrane related trafficking, both endocytosis and exocytosis. many of these processes correspond to key housekeeping functions, explaining the enrichment for essential genes evident in figure 6b. whether the increased translational efficiency of these housekeeping genes following depletion of eif4g is a consequence of relief from translational repression exerted by eif4g, or if it corresponds to a more general cellular effort to counter the effects of loss of eif4g, is not clear. notably, the genes translated less efficiently following depletion of eif4g tended not to encompass essential genes, and several housekeeping processes, such as dna processing and protein modification were underrepresented in this group. in contrast, it was enriched for genes involved in oxidative stress response, especially components of the cellular peroxidase/thioredoxin systems, such as gpx hyr trx srx and tsa these findings suggest that under conditions of eif4g down-regulation, a select group of mrnas whose products function in housekeeping processes such as transcription and dna processing, are translated relatively better than all other mrnas; whereas a group of non-essential genes involved in cellular energy production are translated relatively worse. given the reported involvement of eif4g in activating mrnas for recruitment of the 43s pic and scanning the 5'utr, we examined the two sets of genes with significantly altered te4g/tewt ratios to determine whether they exhibit atypical 5'utr lengths or secondary structures. we employed the database of 5'utr lengths for yeast orfs from lawless et al compiled from results of genome-wide studies of 5' transcription start sites. interestingly, for the genes with te4g/tewt ≥ whose features were compiled by lawless et al, the mean 5' utr length is ± nt, which is ~ fold greater than the average 5'utr length of ± nt for all genes in the database. for the genes with te4g/tewt values ≤, the mean 5' utr length is ± nt, significantly smaller than that determined for the genes with te4g/tewt ≥ but not significantly different than the mean value for all mrnas. the enrichment for long 5'utr lengths for genes with te4g/tewt ≥ is evident in figure where their length distribution is compared to that of all 5'utrs. thus, the fraction of genes exhibiting a relative increase in te in the mutant have a significantly longer than average 5'utr, whereas those exhibiting a relative decrease in te on eif4g depletion have a nearly typical length distribution. thus, the class of mrnas most dependent on eif4g exhibit the comparatively short 5'utr lengths characteristic of the majority of yeast mrnas. using the computer program randfold to predict folding of 5'utrs, lawless et al reported that the vast majority of yeast 5' utrs appear not to be strongly folded, with only 5'utrs showing low minimum free energies of folding with an associated p-value of <. none of these mrnas was found among the genes showing the greatest reductions in te in the mutant versus wt. in fact, four of these mrnas, all containing long 5'utrs with strong predicted secondary structures, appear to be translated more efficiently on depletion of eif4g, showing mean te4g/tewt ratios of ±, ±, ±, and ±. these results do not support the possibility that the translation of mrnas with highly stable secondary structures in their 5'utrs would be strongly enhanced by eif4g. it has been reported that mammalian eif4g plays a critical role in the ability of post-termination 40s subunits to resume scanning following translation of a short uorf. hence, we asked whether the genes whose translation is relatively lower in the mutant versus wt might display an atypical occurrence of uorfs. for the genes with te4g/tewt values ≤ whose occurrences of uorfs were tabulated by lawless et al, there is an average of ± uorfs per transcript. for the genes with te4g/tewt ≥, the corresponding average is ± uorfs per transcript. neither of these frequencies differs significantly from the average uorf occurrence of ± uorfs per transcript tabulated for genes by lawless et al. thus, we found no indication that the presence or absence of uorfs is a critical determinant of the effect of eif4g on the translational efficiency of eif4g-responsive mrnas. in animals, translational control of specific mrnas frequently involves trans-acting factors that bind to specific recognition elements in the 3'utr and target eif4f assembly at the cap structure. accordingly, we examined whether the 3'utr length differs significantly between the two sets of genes identified above. as shown in figure the 3'utr length appears to be slightly smaller for the group of genes with te4g/tewt ≤ versus that with te4g/tewt ≥; however, neither group displays a mean 3'utr length that is significantly different from that of all genes. hence, it seems unlikely that 3'utr length is an important parameter in determining the dependence of translational efficiency on eif4g. finally, we examined mrnas reported to have an a-rich ires and also the ires-containing mrna ure to determine whether the translational efficiencies of these mrnas might be increased or decreased on depletion of eif4g. we observed no significant deviation from unity in the te4g/tewt ratios of the genes with a-rich iress: ±, ±, ±, ±, ±, ±, ±, ±, ±. the same holds true for the ure2/ynl229c mrna, exhibiting a te4g/tewt ratio of +. these findings provide no indication that eif4g is a critical factor for the function of these ires elements, although it is unknown whether any of the iress are functional under the nonpermissive growth conditions of our experiments. most genes with short orfs require eif4g to achieve their characteristic, higher than average translational efficiencies because we examined polysomal rnas present in "heavy polysomes", genes whose transcripts contain, on average, less then translating ribosomes were likely underrepresented in this analysis. of particular concern are those genes with relatively long coding regions whose mrnas have an average of or fewer translating ribosomes in wt cells, i.e., with low ribosome densities, because such inefficiently translated mrnas might be particularly dependent on eif4g. to address this possibility, we extended our microarray analysis to include rna isolated from the "light polysome" fractions obtained from the same gradients that yielded the hp fractions analyzed above. the mean te for each gene was calculated as the ratio of lp/t rna intensities in all three projects, as above for hp rna. we then cross-referenced the resulting te values with a database listing the ribosome densities of yeast genes described by arava et al., focusing on a group of genes whose mrnas in that study displayed peak occupancies of only 1- ribosomes per mrna, and thus should occur primarily in the lp fractions of our study, of which were interrogated on our microarrays. a subset of genes from this group contain relatively long coding sequences and exhibit average ribosome densities of ≤ ribosomes per nt-well below the genome-average density of. the mean tewt calculated for these genes from our lp data, +, is significantly below the genome average tewt value of ± derived from the hp data for all orfs, indicating that these genes exhibit an atypically low proportion of mrna associated with ribosomes in addition to a low ribosome density. consistent with our findings on mrnas in hp fractions, the majority of these poorly translated mrnas in the lp fractions exhibit higher te values in the eif4g mutant versus wt cells. thus, it appears that eif4g is not a critical rate-limiting factor for this group of very inefficient mrnas. we also examined a subset of genes from the group of mentioned above, which exhibit peak occupancies of only 1- ribosomes per mrna simply because they have short orf lengths, as their mean ribosome density actually exceeds the genome-average of. interestingly, these genes have a mean tewt value of ±, that is substantially higher than the genome-average tewt value and most of these genes have significantly lower te values in the eif4g mutant versus wt. having identified a group of efficiently translated mrnas with a marked dependence on eif4g that contain atypically short coding sequences, we examined the behavior of all genes with short orfs in both the lp and hp data sets. as illustrated in the log-log plots of figure s, ~90% of these genes exhibit te values greater than unity in wt cells, compared to only 55% for genes of all orf lengths. this disparity reflects the broader phenomenon that tewt values are inversely related to orf length, as revealed in the scatterplot of tewt values versus orf length for the entire hp dataset. this relationship is not unexpected, as it was noted previously that ribosome densities on mrnas and protein expression levels are inversely related to orf length in yeast. interestingly, the majority of the short-orf genes exhibit reductions in te values in the eif4g mutant of ~10% on average, similar to the average reduction in te mentioned above for all genes with tewt values above unity. the reduction in te evoked by depletion of eif4g for small-orf genes is also obvious in the scatterplots of figure s, as dampening te values for the shortest orf lengths in the eif4g mutant is observed. thus, genes with short orfs tend to be translated more efficiently in wt cells and to be dependent on eif4g for their maximum efficiency. it is noteworthy that the two sets of ~ genes we identified above displaying the greatest changes in te values on depletion of eif4g differ dramatically in average orf length. the group exhibiting the greatest reductions in translation efficiency has a mean orf length below the genome average by nearly a factor of two, while genes showing the greatest increases in efficiency have a mean orf length 70% larger than average. these findings suggest that orf length, in addition to 5'utr length, determines the influence of eif4g on translational efficiency. below, we propose a molecular explanation for this finding, based on the known relationship between transcript length and the stability of eif4f-cap interaction. considering the strong correlation between orf length and effect of eif4g depletion on translational efficiency shown in figure it seems possible that the enrichment of cellular functions associated with the gene sets exhibiting te4g/tewt ≤ or te4g/tewt ≥ described above could at least partially reflect a preponderance of genes with unusually small or large orf lengths in those functional categories. discussion in this study, we have examined the genome-wide consequences for translational efficiency of simultaneously eliminating eif4g and depleting eif4g from yeast cells. the conditional depletion of eif4g achieved using a degron-tagged version of this protein was highly effective and reduced the polysome content and rate of translation to only 20-30% of wt levels, indicating a substantial reduction in the rate of translation initiation. we used genome expression microarrays to measure the abundance of each mrna in heavy polysomes relative to its level in total mrna to calculate translational efficiencies of different genes. the results indicated that the overwhelming majority of mrnas experienced only a moderate change in translational efficiency on eif4g depletion. less than 2% of the genes showed a statistically significant decrease in te in the mutant by a factor of of more, and the genes in this group that were affected the most displayed reductions of a factor of ~ or less. while the actual percentage of genes affected to this extent is probably higher, only ~10% of genes exhibited decreases in te of this magnitude for each biological replicate, which likely represents the upper size limit for this category. thus, we did not detect even a small group of mrnas that are dramatically dependent on eif4g for translation in vivo. we made the unexpected observation, however, that depletion of eif4g narrows the range of translational efficiencies for a large fraction of mrnas, decreasing the number with efficiencies that are substantially higher or lower than the genome-average te. this trend is well illustrated in the log-log plots of mean te values in wt versus mutant cells, and also by the fact that depleting eif4g reduced the numbers of mrnas with te values either -fold higher, or -fold lower, than unity. furthermore, the bulk of mrnas with te values ≥ in wt cells are, at least to some extent, dependent on eif4g for their higher than average te values. this dependence is consistent with a significant role for eif4g in stimulating one or more steps of initiation for the most efficiently translated mrnas in the cell, presumably the activation of mrna for recruitment of the 43s pic, scanning the 5'utr, or start codon recognition. unexpectedly, we found that many mrnas with lower than average te values in wt cells exhibit an increased translational efficiency on eif4g depletion. it is conceivable that eif4g directly impairs the translation of these latter mrnas. however, we favor an indirect mechanism involving competition among all mrnas for limiting initiation factors or pics, coupled with the role of eif4g in stimulating efficiently translated mrnas at the expense of those with lower than average efficiencies. in the absence of eif4g, this competitive edge would be eliminated for the first group and thereby enable the second group of mrnas to compete better for limiting factors/pics. the small group of ~ genes we identified that are most dependent on eif4g for their higher than average tes in wt cells contain a mean 5'utr length that is slightly below the genome-average for all mrnas, a feature that should facilitate efficient scanning and aug recognition. this was surprising because we expected to find that the mrnas most dependent on eif4g would have long or highly structured 5'utrs, requiring the eif4g·eif4a complex for unwinding secondary structure to promote 43s attachment or scanning. in fact, the ~ genes we identified whose translation is stimulated the most by eliminating eif4g contain a mean 5'utr length substantially larger than the genome average. the fact that these latter mrnas display a lower than average te in wt cells and benefit from the absence of eif4g seems to indicate that they function inefficiently at steps of initiation not significantly enhanced by eif4g. given their long 5' utr lengths, it seems likely that scanning to the start codon is relatively inefficient for these mrnas. if so, then the fact that depleting eif4g does not exacerbate this deficiency suggests that factors besides eif4g are more critically required for efficient scanning through long 5'utrs in yeast. this last suggestion is consistent with our finding that none of the mrnas predicted by the randfold program to contain the most stable secondary structures among yeast 5' utrs displayed a significant reduction in te on eif4g depletion--in fact, four such mrnas appear to be translated more efficiently on eif4g depletion. thus, other initiation factors besides eif4g might also be more critically involved in removing secondary structures in advance of the scanning pic. this view is supported by the fact that in a mammalian reconstituted system, eif4g, eif4a and eif4b are sufficient for 43s attachment and scanning on β-globin mrna, which harbors a relatively unstructured 5'utr, whereas the dexh-box protein dhx is required for initiation complex assembly on mrnas containing more structured 5'utrs. similarly, there is evidence that yeast dead-box protein ded contributes more than eif4a does to the processivity of scanning in vivo. these findings are in agreement with the possibility that the eif4e/eif4g/eif4a complex is more critical for 43s pic attachment near the 5' end of the mrna than for subsequent scanning to the start codon. thus, our results are consistent with the model that 43s attachment is a rate-limiting step for a large proportion of mrnas with higher than average tes, and that this step is stimulated by eif4g, particularly for the ~ genes we identified with the greatest dependence on eif4g that contain relatively short 5'utrs. by contrast, scanning or aug recognition would be rate-limiting for mrnas with longer than average 5'utrs whose translation is enhanced by depletion of eif4g, because these steps are not critically dependent on eif4g. the fact that eliminating eif4g mitigates the lower than average translational efficiencies of this second group of mrnas can be explained by proposing that the negative effect of depleting eif4g on 43s attachment is outweighed by their enhanced ability to compete with other mrnas for limiting factors that promote scanning or aug recognition. fulfilling this last stipulation of our model would be facilitated if the inefficient mrnas with long 5'utrs are relatively ineffective at exploiting eif4g function in 43s attachment. that is, if eif4g contributes relatively less to 43s attachment by these inefficient mrnas in wt cells, then depleting eif4g would produce relatively smaller reductions in their translation rate. one reason for thinking that this condition holds is our finding that this group of mrnas also displays unusually long coding sequences, whereas the mrnas we identified with the greatest dependence on eif4g exhibit smaller than average orf lengths. recent findings by jacobson et al indicate that shorter yeast mrnas produce more stable eif4f-cap interactions than do longer mrnas, which is fully dependent on an extended poly tail and pabp. presumably, shorter mrnas more efficiently assemble a closed-loop mrnp via pabp-eif4g interaction, which stabilizes eif4f binding to mrna. in fact, the possibility of less efficient 5'-3' interaction for larger mrnas was advanced previously as one explanation for the inverse correlation between ribosome density and orf length, which we confirmed here using te values. hence, we suggest that longer mrnas are affected less than shorter mrnas by the elimination of eif4g because the eif4f-cap interaction is inherently less stable for longer transcripts and, hence, less efficacious in promoting 43s recruitment when eif4g is present. the fact that depleting eif4g diminishes, but does not eliminate the correlation between te and orf length indicates that reduced eif4g-pabp interaction is not the only factor limiting the translation of mrnas with longer orfs, and limited processivity of elongating ribosomes or less efficient termination have been suggested as other possibilities. we showed previously that depletion of eif4g did not lower the amounts of native 48s complexes containing the rpl41a or mfa mrnas, both very short transcripts, which is ostensibly at odds with the idea that eif4g has an important function in 43s attachment to mrna. examining the results we obtained for these mrnas in the lp dataset reveals that they both exhibit mean te4g values ~90% of their tewt values. thus, even if we assume that these two mrnas require eif4g only at the step of 43s attachment to achieve their maximum translation rates, it would have been very difficult to detect a 10% decrease in the levels of their free 48s complexes with the techniques employed in the previous study. it remains to be determined what features in mrna, besides a short 5'utr and short orf length, are responsible for the more pronounced requirement for eif4g displayed by the small fraction of yeast mrnas identified here. considering that eif4g is essential in yeast, and also noting its role as a protein bridge linking the eif4e-mrna-pabp mrnp to components of the 43s complex, it is surprising that a significant amount of translation still proceeds in the absence of this factor. based on our microarray data, it appears that eif4g is dispensable for the translation of most, if not all mrnas in vivo, indicating that it is rate-enhancing rather than essential in budding yeast. this stands in contrast to the critical requirement for the eif complex, which is required for nearly all translation in yeast, and is crucial for attachment of native 43s complexes to mrnas that can assemble 48s pics in cells depleted of eif4g. of course, we cannot exclude the possibility that a compensatory initiation pathway comes into play during the h of incubation in the non-permissive conditions used to thoroughly deplete eif4g. it is also impossible to eliminate the possibility that a very small fraction of the wt amount of eif4g, below the detection limit of our western analysis, is sufficient to catalyze the residual protein synthesis that occurs in the depleted cells. this seems unlikely, however, because the eif4g level in wt cells is already lower than those of nearly all other initiation factors. on the other hand, the to 4-fold reduction in the rate of translation, and the narrowed range of translational efficiencies evoked by depletion of eif4g, could have serious consequences for a subset of dosage-sensitive proteins with essential functions in the cell. moreover, cell division could be blocked under these conditions by regulatory mechanisms that respond to a drop in the rate of synthesis of a key cell cycle controlling factor, eg., the g cyclin cln. considering that cell division is not blocked by a decrease in the overall translation rate of ~70% occurring in response to hyperosmotic stress, eif4g depletion might evoke a comparatively greater reduction in translation of a key protein required for cell division than occurs during osmotic stress. given that depletion of eif4g reduces the translation rate by to 4-fold, it is surprising that the average te calculated for all genes decreased only a small amount, from ± in wt cells to ± in the mutant. of course, many genes translated with higher than average efficiencies in wt exhibit much larger reductions in te values on depletion of eif4g, but this effect was counterbalanced by increased translation of many genes with lower than average tewt values. as noted above, the fact that microarray results are normalized to give each array the same average signal intensity will dampen the reduction in polysomal mrna abundance in the eif4g mutant, and the amounts of total mrna might also decline on eif4g depletion, which would offset the effect of decreased polysomal mrna on the calculated te values. it is also conceivable that eif4g depletion triggers a signal transduction response that decreases the rate of elongation, counteracting the effect of reduced initiation on polysome size. for example, oxidative stress reduces the rates of both initiation and elongation in yeast. because we examined cells lacking eif4g and depleted of eif4g it could be argued that the changes in translational efficiencies we observed result primarily from the absence of only eif4g or eif4g rather than the elimination of both eif4g isoforms. this is unlikely in view of recent findings by clarkson et al on mutant strains expressing only eif4g or eif4g and engineered to express each isoform at a level equivalent to the combination of both isoforms in wt. these strains displayed almost no changes in translational efficiency genome-wide, providing strong evidence against the possibility that eif4g or eif4g is specifically required to support the translation of particular mrnas. in this same study, two groups of protein-coding genes displayed a significant change in translational efficiency on deletion of only tif encoding the major isoform, which reduced the growth rate and polysome content relative to the isogenic wt strain. only 10% of the genes with significantly repressed translational efficiencies in tif4631Δ cells thus identified by clarkson et al belong to the group of ~ genes we identified here with mean te4g/tewt ratios of ≤. however, the group of translationally repressed genes in the clarkson et al study displayed an average te4g/tewt ratio in our experiments that is significantly below the genome-average te4g/tewt ratio and also the average te4g/tewt ratio determined in our experiments for the group of translationally enhanced genes identified by clarkson et al. thus, the translational efficiencies of at least a subset of genes are affected similarly by the absence of eif4g alone and the elimination of both eif4g and eif4g simultaneously. this is consistent with the conclusion that eif4g and eif4g perform essentially identical functions. a recent analysis of the consequences of depleting eif4gi and eif4gii with sirnas in cultured mammalian cells reached certain conclusions congruent, and others that seem to differ, from our findings. it was found that depleting both eif4gi and eif4gii reduced overall translation by only ~20%, whereas depleting two eif subunits provoked a stronger reduction, consistent with the greater requirement for eif versus eif4g we observed in yeast. eif4gi depletion reduced the translational efficiencies of a subset of mammalian mrnas, including a group whose products function in mitochondrial regulation, bioenergetics, and cell proliferation. in accordance with our observations, there was no significant correlation between the presence of long or structured 5'utrs and the degree of eif4gi-dependence. this is consistent with the aforementioned suggestion that eif4gi is more important for 43s attachment than for subsequent scanning through the 5'utr. at odds with our results, however, the eif4gi-dependent class of mrnas appeared to be somewhat enriched in those containing uorfs, and the presence of an uorf was shown to increase the eif4gi dependence on translation. one possibility is that the majority of uorf-containing mrnas in yeast do not support appreciable reinitiation in wt cells, as this process has strict requirements for uorf length and cis-acting sequences surrounding the stop codon. in this event, eliminating the potential role of eif4g in stimulating reinitiation would be difficult to detect on a genome-wide basis in yeast.  our results indicate that eliminating both isoforms of eif4g from yeast cells elicits a substantial reduction in the rate of translation initiation that is severe enough to block cell division, but does not evoke dramatic changes in the relative translational efficiencies of the majority of mrnas. rather, we observed a large-scale narrowing of translational efficiencies, including mrnas with higher or lower than average efficiencies, which is expected to disturb the stoichiometry of protein components comprising many cellular pathways and structures. our finding that mrnas with the greatest dependence on eif4g are relatively well-translated, do not contain long or highly structured 5'utr, and also have short coding sequences, is consistent with the idea that eif4f is most critically required to enhance 43s attachment to the mrna 5' end rather than for scanning through long, structured 5' utrs. 
we have tested this hypothesis by determining the effects of genetically depleting eif4g from yeast cells on global translational efficiencies, using gene expression microarrays to measure the abundance of mrna in polysomes relative to total mrna for ~ genes. eukaryotic translation initiation factor 4g is thought to influence the translational efficiencies of cellular mrnas by its roles in forming an eif4f-mrna-pabp mrnp that is competent for attachment of the 43s preinitiation complex, and in scanning through structured 5' utr sequences. surprisingly, the fraction of mrnas most dependent on eif4g display an average 5' utr length at or below the mean for all yeast genes. within these limits, however, depleting eif4g narrowed the range of translational efficiencies genome-wide, with mrnas of better than average te being translated relatively worse, and mrnas with lower than average te being translated relatively better. our results also indicate that eif4g, and the closed-loop mrnp it assembles with the m g cap- and poly-binding factors, is not essential for translation of most mrnas but enhances the differentiation of translational efficiencies genome-wide.
 eukaryotic translation initiation factor 4g is thought to influence the translational efficiencies of cellular mrnas by its roles in forming an eif4f-mrna-pabp mrnp that is competent for attachment of the 43s preinitiation complex, and in scanning through structured 5' utr sequences. we have tested this hypothesis by determining the effects of genetically depleting eif4g from yeast cells on global translational efficiencies, using gene expression microarrays to measure the abundance of mrna in polysomes relative to total mrna for ~ genes.  although depletion of eif4g is lethal and reduces protein synthesis by ~75%, it had small effects on the relative te of most genes. within these limits, however, depleting eif4g narrowed the range of translational efficiencies genome-wide, with mrnas of better than average te being translated relatively worse, and mrnas with lower than average te being translated relatively better. surprisingly, the fraction of mrnas most dependent on eif4g display an average 5' utr length at or below the mean for all yeast genes.  this finding suggests that eif4g is more critical for ribosome attachment to mrnas than for scanning long, structured 5' utrs. our results also indicate that eif4g, and the closed-loop mrnp it assembles with the m g cap- and poly-binding factors, is not essential for translation of most mrnas but enhances the differentiation of translational efficiencies genome-wide. 