Supporting InformationPeil et al. 10.1073/pnas.1310642110

Fig. S1. Correlation for biological replicates of proteomics data from Δefp, ΔyjeK, ΔyjeA, and ΔyfcM strains. Scatter plots showing correlation of invertednormalized heavy/light (H/L) ratios (log2-transformed) for biological replicates of SILAC (stable isotope labeling by amino acids in cell culture) data from theΔefp, ΔyjeK, ΔyjeA, and ΔyfcM strains.

Fig. S2. Scatter plots of proteomics data from replicate 2 of Δefp, ΔyjeK, ΔyjeA, and ΔyfcM strains. (A–D) Scatter plots of inverted normalized H/L ratios (log2-transformed) relative to the summed up protein intensity for a biological replicate of SILAC data from the Δefp (A), ΔyjeK (B), ΔyjeA (C), and ΔyfcM (D) strains,including density plots showing distributions of PPP-containing (gold) and XPP/PPX-containing (blue) proteins relative to all proteins (gray).

Peil et al. www.pnas.org/cgi/content/short/1310642110 1 of 8

Fig. S3. Gene Ontology (GO) analysis of complete proteomics data from Δefp, ΔyjeK, ΔyjeA, and ΔyfcM strains. Heat map representation of hierarchicalclustering of all GO terms that are up-regulated (top 25% quantile) or down-regulated (bottom 25% quantile), or remain unchanged (25–75% quantile). Colorkey as indicated: Gray indicates fewer than three genes; white indicates a P value of >0.05; different shades of red indicate increasing significance of P values,from salmon (<0.05) to red (<0.00005).

Peil et al. www.pnas.org/cgi/content/short/1310642110 2 of 8

efp

-1

efp

-2

yje

K-1

yje

K-2

pox

A-1

pox

A-2

yfc

M-1

yfc

M-2

efp cadA pyrC rnb valS atpD plaP ycgR atpC fabF atpF atpG uvrA yjjK pdxB lepA ygdH yhgF bamD nadC mqo malP malQ acnB appC glsA1 pncA adhE ompF speD rpoS poxB lysU hemH hslU tyrB mrp tdh uxuA yeiG hisB ndk glnA yliF ppc cbpA rsmA tpiA lon alaS fkpA gcd trmA pgm clpB fhuA efeO sdaC mnmE sdaB thrB gltD cspC mltA emrA speG frmA codA hisH hsdS yecJ nrdA tsx purN atpH acrB truB trmB atpA qorA evgA alaA asnS galU parC rpoB accD map rlmL aroC mrdA rluB yajG mreC rfbB yiaD rluC dapE yfaY ycfD rfaF cptA iadA lamB manX malE pyrD dps gadA hdeA hdeD slp gatZ gatA gatC recB yfdI yfhM galK galE purA ndh fadL copA cspA osmY ybaY acnA otsA ampC hemY xseA glgB fliS hisC hisG pal pyrF pncB ydgH thrA thrC gltB tgt cfa fruR corC hexR yfhL speE mrcB mipA tolA rimM bglX dapB tyrA mprA era rmlA1 ybgF nuoI hisA ompC ybeB speA ycgL clpA iciA dsbC zwf mltB rpmE rplJ rplT rpsB rpsC rpsJ rpsM rpsF rpsI rplL rplO rpmA rplF rpmD rpmG rpsH rplX rplS rpsG rpsS rplV coaA rpmC yeiP rpsQ rplA rpsK rplM rplC rplD fabH metJ yheS glnS rlmI rpmH rimO yedY cyaA infB thrS rpmF glmM srmB secF lepB rseP deaD ansA rplI rpsR rplR rpsD rplP rpsN nusA rplE rplY rpsL rplU yggS rpsT rpsE rplN rplQ rdgB jayE uup aroK rpsO damX glf btuB dsbG def fis yeaD accA fmt ydgA aceF sucB pyrH sucA pitA rsmB nagC mdoB metL kdsB ispB serS ybhC gpsA zur accC yihI kbl yjaG ftsK rpsU tatC gltX parE rplK pcnB rplB rplW rpsA lpxD nupC wzzE rcsD purK fabB purM queE hsdM prfC yeeN murI rhlE secD ffh thiI yacF ygdL yibL stpA nrdB cyoB exbB speB hsdR aceE nuoC nuoG pgi yncD folE ybiV metK wecC rfbD pldA yefI rfbC rsmG rffG potA rlmG infA sodB glnP glnQ yjiA sppA ygdI maeA ptsG ybjX flhA dut gcvH orn coaBC mutS pgk glyA guaA pykF eno fabA yfiF hisJ yphE folP gsk purF purL potD sodA ycbZ gpmI gshB livK ybaB frdA carA carB ucpA leuS glk lpdA sdhA fbaA pepQ yfcZ accB der plsB engD hpt fabD fabG trmJ fabZ atpE pfkA ppiC purB hldE rnt cmoB rfaS ycfH purC purE bioA sdhB yidC queA ribH waaU ppa ubiE surE rfaG fre seqA topA ppiA usg ftsY hflK hisS rhlB rsmC glmU argS hldD sthA yigL hemE fbp cyoD xthA ftsZ frr yegQ rng rpsP proQ folC nusB gpmB yaaA rfaH gpt prlC fdhE rsgA udk guaC dtd ftsX rnc lptG rsmI yacG yefG rbfA nhaB modA ptsI tktA lysS prfB tsf rlmN rpoZ slyD mpl gshA tig rne cmk secA truD mrcA queD obgE ydcP proP proS mfd recC nuoF rna cyoA erpA yhiI acpP bamA nudF surA yicC yccF ybhA rpmB tufA;tufB dxs pdhR pepD htrB secY yqaB corA nagZ proB nudC amiC wzzB helD miaA ygaD ftsE ptrA ygiC ompT rseB yffB dapD lipA ygiF creA dmsD pth pheT ptsH pyrG gcp gyrB fabI polA spoT hemX pmbA hemL rpoC panB yaiL rho mnmA nagD ycdY msbB pnp pheS sseB secG rlmF yidA tolC dapA ygfB ftsH murC rpoN ompA murF fusA nfuA dacA yhbY ribF purR gsp ispG aroE rimN murE yebT yfgD slyB ydcY ybiS ddlA hemG mukB coaD rfaB kdsC dnaB fldB ygdK yibN ompX hflC folD mtnN cysS yfgM rep gapA selB rfaI kefA gmk dnaT ahpF cspE tehB pepN ligA nlpI lolB mdoD yecD pdxH rsuA prc ydfG ptsP htpX tolQ yiaF mukF recA glrR mreB rodZ nanR lptD diaA csrA yhcM mgtA yfiH mukE suhB bioB pepB nuoE hmp katG betB nmpC cysQ purH purD rapA ycaO rbsD ppsA glnD dld fliA nadB talA yeaG moaC yceI tsr tar mdaB cheA cheW flgH motA motB pyrB pyrI oppC relB aphA oppB groL htpG narP ybiT yejF oppD gor nfo yfbU ycfP yacL deoD pckA nrdR ybaL aroA yggE nadR elbB arcA sdaA zapB yggL yhbS ribB rlmE yraP tmk cysB aroD rcsF ybgI minE cobB hns pgl ypeA ldcA trpA proC prmB narL nadE ybjP dacC ackA yeeX frsA nfsA murA clpP icd luxS hscB iscS nifU grpE pykA yeeZ msrB purU yccU hemB ssb slt tyrS gyrA oppF yhbJ yifE pdxY gph glyQ mlaC ribE ppiB rpiA ispF ribD queF fldA crr elaB yiiM yqjD ygiM mdoG ghrA qorB glgA ldhA gltL cysZ ppsR fbaB yciN ygiN yadG trpB lrp ydjA metH metQ gnsB yghU yggX glnE dnaJ groS ybbN ghrB oxyR grxD uspA ymbA yceH tesB yecS yqhD mdh moaE wbbL lpxC tas kdsA sucC sucD dksA mscM deoC ppk tdcF holE aspS cpdB moeA fdx yhcB ppiD hscA selA ung grxC can pta bioH artI msbA hslV yceD cls yedD rpoD apt greA menB dnaA pssA folB ddlB lptB bamC acrA hslO hupA bamB hfq yajC fklB kdgR fkpB nuoB nusG nagB pcm yqiC eda gloA secB lpoA rlmB gpmA ychN ushA yebC fur trxB sspA yhhX dnaK fumA ybeZ manA talB ridA gmhA yejK rcsB galF miaB dinJ typA pdxJ sohB ltaE minC rcnB infC prs gnd prfA rpoA psd hdhA cysE yihD modE pdxK yiaJ pflA nlpD trxA trpS hemN lptA yhdH yeiE mlaA clpX yoaB selD ygfZ yajL smpB metN ugpQ baeR ybfF deoB ycaC eco degP upp asnB tyrR proA arcB nfnB ubiG dcp ydiJ ilvI yajD dhaM ydiI yciM ybaK allR pntA panD zipA glnB skp hinT folX ybcJ yniC metG cydC lpp hflX yfbT yebY ycbL ompR serC yrdA lexA adk phoP aroB tolB iscA minD ytfP bcp tpx panC nudE ruvB yddE ahpC yfcE pntB yggG degQ crp crl aroG hupB lolD msrC ynfB yieF mscS solA yceA rsmH fliM fliG fliL ispH yeaK tabA guaB dsbA phnA purT yjgA katE engB ubiF argD ileS cpxR ydhF rnr pepA galM lpxA avtA glyS dcrB thyA rob tldD mlaD murB rdgC relA yajQ bglA glmS yhiR cmoA trkA gcvT yqcC yfeX yjeE thiL kdgK yhjH yrfG tmcA glgP fliN cydA hdeB ycaR glnH pflB ygiW manZ asnA gatY aspA ydbK yqjI borD dadA aspC serA asd yjcD ftnA ivy yeaJ nemA ibpA ydgJ yjgR lysC grxA metE azoR hrpA ilvE pstS leuC leuD leuA pheA leuB phoB sra ftsA phoU pstB yciI cheB cheZ cheY aceB gstA moaB btuE ydcL osmC udp dcyD cdd ihfA ihfB hpf rpe iraP cysK gstB ycbX amn fliY gltI yaeH sbmC gdhA ybiC fliZ fliC fumC gcvP uspD rraA alaC wrbA yahK yccJ potF rbsB yjbR glgC oppA maeB grxB pepT gltA yajO flgK flgG flgE flgD osmE ecnB argG argH argE yhaM mtlD flgB tap putA glpK aceA gabT hchA ygaU bfr gabD dppA yjbJ pspA ydhR sseA msyB yeaO flgL uspG znuA flu ilvD ilvB ugpB raiA ilvC lsrF mtlA glcG mglB agp dkgA yniA artJ yahO aldA argT uspF ynaE;ydfK tnaA acs ytfQ

Fig. S4. Hierarchical clustering of complete proteomics data from Δefp, ΔyjeK, ΔyjeA, and ΔyfcM strains. Heat map representation of hierarchical clus-tering of all proteins that are up-regulated (red) and down-regulated (blue) in the biological replicates of Δefp, ΔyjeK, ΔyjeA, and ΔyfcM strains relative towild-type strain.

Peil et al. www.pnas.org/cgi/content/short/1310642110 3 of 8

Fig. S5. Box plot representations of inverted normalized H/L ratios (log2-transformed) for proteins containing XPP motifs for Δefp, ΔyjeK, ΔyjeA, and ΔyfcMstrains.

Peil et al. www.pnas.org/cgi/content/short/1310642110 4 of 8

Fig. S6. Box plot representations of inverted normalized H/L ratios (log2-transformed) for proteins containing PPX motifs for Δefp, ΔyjeK, ΔyjeA, and ΔyfcMstrains.

Peil et al. www.pnas.org/cgi/content/short/1310642110 5 of 8

Fig. S7. Box plot representations of inverted normalized H/L ratios (log2-transformed) for proteins containing RME, YIR, PFF, and GPG motifs for Δefp, ΔyjeK,ΔyjeA, and ΔyfcM strains.

Peil et al. www.pnas.org/cgi/content/short/1310642110 6 of 8

Fig. S8. Translation of non-XPPX motifs in the absence of elongation factor P (EF-P). (A) β-galatosidase activities (normalized in wild-type Escherichia colistrains relative to Δefp strain) of LacZ constructs containing control (no stalling motif), PPP, or PPG, compared with LacZ constructs containing YIRYIR or PoxBmotifs GSCGPG or GSC. (B) Autoradiographs of SDS–polyacrylamide gels indicating [35S]Met-labeled in vitro translation of reporters containing (lanes 1–6) RME,PFF, YIR, GPG, GSCGPG compared with PPP. Reactions were performed in the absence (−) of active EF-P. Expected peptidyl-tRNA and full-length (FL) productsizes are indicated. The bar graph presents the relative stalling, which is calculated as the amount of stalled peptidyl-tRNA product/(stall + FL product). Allrelative stalling values are presented relative to that determined for PPP, which was assigned as 100%.

Peil et al. www.pnas.org/cgi/content/short/1310642110 7 of 8

Table S1. Kruskal–Wallis significance for protein ratio distribution differences betweenthe motif subsets and complete dataset

Motif efp-1 efp-2 yjeK-1 yjeK-2 yjeA-1 yjeA-2 yfcM-1 yfcM-2

PPP 1.31E-04 1.80E-06 7.37E-05 5.70E-08 2.81E-05 1.08E-04 3.98E-01 9.78E-01APP 3.31E-06 1.94E-08 9.03E-08 2.99E-07 5.71E-06 8.24E-05 2.02E-02 5.76E-02CPP 1.64E-01 1.26E-01 1.98E-01 9.46E-02 3.32E-01 3.12E-01 8.68E-01 5.63E-01DPP 1.03E-03 1.03E-06 1.76E-07 2.37E-06 8.33E-04 2.96E-04 1.03E-01 3.01E-01EPP 3.04E-02 1.93E-01 2.81E-01 1.24E-01 3.91E-01 5.81E-02 7.00E-01 5.91E-01FPP 8.91E-01 9.71E-01 5.59E-01 6.53E-01 6.58E-01 4.52E-01 1.53E-01 9.89E-01GPP 1.77E-01 1.64E-01 7.87E-03 7.55E-03 9.49E-02 1.63E-01 2.55E-01 8.24E-01HPP 3.65E-02 1.16E-01 1.88E-01 2.08E-01 2.33E-02 3.83E-02 7.05E-01 2.23E-01IPP 3.85E-02 8.82E-04 4.19E-05 3.92E-05 1.64E-02 4.01E-02 1.32E-01 6.38E-01KPP 2.31E-01 2.05E-01 3.49E-02 6.41E-02 3.71E-01 1.69E-01 6.86E-01 6.75E-01LPP 9.09E-01 5.05E-01 1.88E-01 1.04E-01 7.22E-01 2.85E-01 5.83E-01 6.77E-01MPP 8.03E-01 1.53E-01 4.62E-01 7.37E-01 4.98E-01 2.54E-01 6.00E-01 9.15E-01NPP 5.43E-01 5.86E-01 3.19E-02 2.31E-01 3.52E-01 5.09E-01 2.37E-01 1.80E-01QPP 5.30E-01 4.84E-01 3.04E-01 3.57E-01 9.53E-01 4.29E-01 4.44E-01 5.68E-01RPP 6.73E-01 9.20E-01 3.23E-01 2.37E-01 8.33E-01 6.17E-01 7.97E-01 1.48E-01

PP 2.35E-01 7.09E-03 1.22E-02 1.69E-03 2.15E-01 3.54E-01 7.86E-01 7.16E-01TPP 8.06E-01 6.78E-01 8.13E-01 4.88E-01 9.96E-01 6.61E-01 7.30E-01 1.95E-01VPP 6.27E-01 7.82E-02 1.75E-02 1.09E-02 1.82E-01 2.65E-01 8.77E-01 7.57E-01WPP 5.30E-02 4.22E-01 1.99E-01 2.87E-01 4.95E-01 7.41E-01 6.48E-01 7.70E-01YPP 2.54E-02 4.68E-01 8.08E-01 7.24E-01 5.61E-01 1.46E-01 5.54E-03 2.65E-02PPA 6.12E-01 1.84E-01 5.72E-02 6.37E-02 3.54E-01 7.63E-01 3.24E-01 9.70E-01PPC 9.98E-01 3.42E-01 7.57E-01 5.93E-01 2.87E-01 4.64E-01 3.06E-01 8.77E-01PPD 5.83E-01 6.67E-01 2.95E-01 2.09E-01 7.05E-01 4.10E-01 1.64E-01 4.41E-02PPE 3.36E-01 2.22E-02 7.82E-02 1.51E-01 1.32E-01 4.84E-02 2.96E-01 8.68E-01PPF 3.56E-02 3.41E-01 3.89E-01 2.22E-01 1.42E-01 1.45E-01 8.43E-01 6.54E-01PPG 3.71E-03 8.62E-07 1.80E-08 1.03E-08 7.12E-04 1.60E-04 6.33E-01 2.11E-01PPH 1.10E-01 4.75E-01 6.22E-01 3.60E-01 8.10E-01 7.15E-01 4.51E-01 2.53E-01PPI 9.70E-01 7.50E-01 4.09E-01 3.81E-01 5.24E-01 9.99E-01 3.89E-01 1.33E-01PPK 1.28E-01 1.14E-01 1.02E-02 3.98E-03 1.60E-01 7.25E-03 9.92E-01 7.19E-01PPL 2.84E-01 2.40E-01 2.37E-02 5.17E-02 4.27E-01 6.96E-01 9.91E-02 3.29E-01PPM 3.25E-01 4.99E-02 9.07E-02 3.46E-02 1.72E-01 1.18E-01 4.56E-01 4.39E-01PPN 8.86E-02 7.93E-04 2.67E-03 7.14E-04 1.57E-02 4.18E-02 1.83E-01 3.05E-01PPQ 4.44E-01 1.89E-01 6.72E-01 4.23E-01 8.71E-01 2.86E-01 3.76E-02 4.53E-01PPR 2.64E-01 3.89E-01 6.44E-02 1.56E-01 8.99E-03 2.53E-01 6.75E-01 4.25E-01PPS 8.38E-01 5.08E-01 7.27E-02 6.62E-02 4.53E-01 3.58E-01 8.93E-01 8.38E-01PPT 4.58E-01 1.18E-01 4.26E-02 1.46E-01 2.22E-01 4.17E-01 9.31E-01 8.04E-01PPV 8.00E-01 5.49E-01 7.15E-01 2.74E-01 4.81E-01 5.73E-01 9.60E-01 6.29E-01PPW 7.00E-01 5.57E-01 5.75E-01 5.68E-01 8.69E-01 5.27E-01 6.09E-01 9.54E-01PPY 5.02E-01 8.76E-01 3.20E-01 5.14E-01 8.09E-01 1.00E+00 9.69E-01 3.14E-01(A/D/I)PP(G/N) 5.24E-05 5.20E-09 2.70E-09 1.30E-09 7.01E-07 2.65E-06 1.05E-01 5.81E-01RME 3.44E-01 1.69E-02 1.04E-02 3.49E-02 2.28E-01 1.01E-01 4.00E-02 1.04E-01PFF 1.64E-01 5.23E-02 4.53E-02 1.15E-02 3.53E-01 5.26E-01 7.81E-01 2.75E-01GPG 4.16E-01 2.39E-01 1.06E-01 1.02E-01 8.36E-01 3.87E-01 5.81E-01 8.56E-01YIR 4.49E-01 8.39E-01 9.21E-01 9.66E-01 8.64E-01 8.54E-01 3.19E-01 7.63E-01

S

Nonparametric Kruskal–Wallis test was applied to test for protein ratio distribution differences be-tween the PPP (orange), XPP (pink), PPX (blue), and non-diprolyl (green) motif subsets and the completedataset (without the corresponding motif subset) for each strain; corresponding P values are listed, withP < 0.05 highlighted in red.

Other Supporting Information Files

Dataset S1 (XLSX)

Peil et al. www.pnas.org/cgi/content/short/1310642110 8 of 8