start site contribute to efficient initiation of translation in vitro

MOLECULAR AND CELLULAR BIOLOGY, May 1991, p. 2656-2664 Vol. 11, No. 50270-7306/91/052656-09$02.00/0Copyright © 1991, American Society for Microbiology

Both the 5' Untranslated Region and the Sequences Surrounding theStart Site Contribute to Efficient Initiation of Translation In Vitro

DOMINA FALCONE AND DAVID W. ANDREWS*Department ofBiochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

Received 24 October 1990/Accepted 10 February 1991

The role of RNA sequences in the 5' leader region between the cap site and initiating AUG in mediatingtranslation was examined in vitro. Hybrid mRNAs were synthesized in which the cognate leader sequence wasreplaced with either optimized or compromised leader sequences, and translational efficiency was measured forsix different coding regions. Translation was most efficient with a leader containing the 5' untranslated regionfrom Xenopus 3-globin and an optimized initiation sequence. Compared with the cognate leaders, this hybridwas observed to increase translation of the various coding regions as much as 300-fold. The translationalefficiencies of the different coding regions also varied substantially. In contrast to earlier suggestions thatincreased leader efficiency results from higher affinity of the leader for a limiting factor, our experimentssuggest that increased translation from the 0-globin hybrid leader sequence results from more rapid initiationof translation.

The relative translational efficiencies of different mRNAsvary widely both in vitro and in vivo (reviewed in references7, 26, and 34). The molecular basis of this variation has beenthe subject of intense scrutiny for many years. On the basisof these studies, several basic features have been identifiedin the 5' untranslated leader ofmRNAs expressed in eukary-otes which lead to efficient translation in vitro and in vivo.Among these are a cap site, an untranslated region (UTR)longer than four to five nucleotides which lacks stablesecondary structure, and a consensus sequence surroundingthe AUG where translation initiates (the start site). The5'-terminal cap structure has been shown to facilitate theinitiation of translation by direct interaction with specificcap-binding proteins (4, 23, 40, 43, 44). However, the role ofthe untranslated sequence between the cap site and the startsite is less well defined. In some specialized systems, thisregion has been shown to contain sequences which areinvolved in regulation of translation (6, 20, 21, 36, 45).The initiation sequence and its position relative to the cap

site determine the selection of a particular AUG codon as thestart site for translation in eukaryotes. In higher eukaryotes,a consensus sequence for initiation has been proposed froma comparison of vertebrate mRNAs (25). Site-directed mu-tagenesis experiments indicate that the most important res-idues of the initiation sequence include positions -3 to +4,with the optimal sequence being (A/G)CCAUGG (24). Thesingle most important residue in the motif is the A or G atposition -3, with an A present in 75% and a G in 20% ofmRNAs. If the nucleotide at the -3 position is not an A orG, then efficient translation requires that there be a G atposition +4 (24). The importance of an A at the -3 positionhas also been confirmed in vivo, since a C at this position hasbeen reported as the putative cause of an a-thalassemia (38).As an approach to generating highly efficient mRNA for

translation of foreign genes, hybrid mRNAs have beensynthesized in which the cognate leader is replaced with onederived from a highly efficient viral or eukaryotic mRNA (5,10-12, 18, 30, 46). However, the results obtained by usingthis approach have been variable, and for those mRNAs that

* Corresponding author.

are translated more efficiently, the mechanism responsible isnot understood (12, 17).To determine whether the translational efficiency of such

chimeric mRNAs is determined primarily by the sequencesimmediately surrounding the initiation site, as proposedpreviously (29), or whether other sequences are also impor-tant, a variety ofcDNAs were constructed in which differentUTRs, initiation sites, and coding regions were linked. The5' UTR of Xenopus ,B-globin linked directly to the initiationsequence ACCAUGG (provided by a synthetic oligonucleo-tide) was used as a favorable UTR and initiation sequence,respectively. As shown below, substitution of the naturalUTR with this composite leader dramatically improved thetranslation of a variety of different coding regions. Ourresults suggest that the higher levels of translation observedwere due to an increased rate of translation initiation.

MATERIALS AND METHODSRestriction endonucleases and nucleic acid-modifying en-

zymes were obtained from Boehringer Mannheim, NewEngland BioLabs, Promega Corp., and Pharmacia LKB.RNA Guard (Pharmacia LKB) was used as an RNaseinhibitor. The protease inhibitors chymostatin, antipain,leupeptin, pepstatin, and aprotinin were from BoehringerMannheim; [35S]methionine was from DuPont-New EnglandNuclear.

Plasmids. Plasmids containing coding regions with thecognate leader sequences were the generous gifts of S.Prusiner, University of California, San Francisco (hamsterprion-related protein [PrP]); J. Capone, McMaster Univer-sity (herpesvirus protein Vmw65); M. Blajchman and R.Austin, McMaster University (human antithrombin III[ATIII]); L. Lauffer and P. Walter, University of California,San Francisco (canine signal recognition particle [SRP]receptor a subunit). These clones were prepared from theoriginal cDNA isolates by using convenient restriction sites;therefore, the cognate leader sequences may not be com-plete. A plasmid encoding chicken pp6f-fsrc (c-Src) was thegift of D. Morgan, University of California, San Francisco.Subsequent plasmids were constructed by using standardcloning techniques (3, 39). The sequence of each of the six

2656

on February 12, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

MEDIATION OF TRANSLATION IN VITRO 2657

different leader sequences used is shown in Fig. 2, withrelevant restriction sites indicated below. For site-directedmutagenesis, a small region of the plasmid was subclonedinto a separate vector, mutagenized, and subcloned into thedesired expression vector, and the region containing themutation was sequenced before use. In other constructionsin which the manipulations might compromise the plasmidsequence, such as blunting the overhanging ends of restric-tion enzyme-digested plasmids by using mung bean nucle-ase, the relevant regions were also sequenced before use. Tofacilitate subsequent cloning steps as well as make thetranslation initiation sites identical, coding regions that didnot already begin with an NcoI site (human ATIII, SRPreceptor a subunit, hamster PrP, and herpesvirus Vmw65)were modified to do so. Complete details of any of theconstructions are available from the authors. Less favorableUTRs were derived from the polylinker regions of commer-cially available plasmids (pGEM and pSP series; Promega)normally used for the expression of cloned cDNAs. Thefunction of the initiation site was impaired by introducing anA-to-C mutation at the -3 position.

Transcription and translation. Plasmids were transcribedby using SP6 polymerase except for human ATIII with thecognate leader, in which case T7 polymerase was used.Transcription reactions were performed as instructed by themanufacturer (Promega). The ion concentrations in the T7transcription products were adjusted to match those of theSP6 products by precipitating the nucleic acids in ethanoland resuspending them in an enzyme-minus-SP6 reaction.For some reactions, plasmids were linearized prior to tran-scription by digestion with a restriction enzyme that recog-nizes a site outside of the coding region. To minimize theeffect of linearization on transcription, the enzymes used forthis purpose either cut blunt or left a 5' protruding end on theplasmid (37). For comparison of the translation of any seriesof RNAs, plasmids were linearized at the same restrictionsite. After transcription, the amount of RNA synthesizedwas measured in a fluorometer. The relative concentration ofRNA was measured from an aliquot of the reaction mixtureby adding it to a solution of ethidium bromide (0.5 ,ug ofethidium bromide per ml in 5 mM Tris-HCl-0.5 mM EDTA,pH 8.1) and measuring the fluorescence intensity of thesample before and after selective digestion of the RNA byRNase A or high pH. A stock solution of tRNA (1.0 mg/ml)was used as a reference standard for all assays. The mini-mum change in the amount of RNA added to translationreaction mixtures to result in a detectable change in proteinsynthesis was 20 fluorescence units, which corresponds to10% of the RNA added to the translation reaction mixtures(data not shown; see also Fig. 4). A change in fluorescence of20 U is approximately 1 order of magnitude larger than theminimum detectable with our device (Sequoia-Turner model450). To permit direct comparison of the amounts and ratesof translation of different RNAs, equal amounts of RNA(within 10 fluorescence units) were added to each reaction,as transcription products. In general, the amounts of RNAsynthesized from the different templates within a singleexperiment were similar enough that concentration adjust-ments were not required. As shown below, replicate inde-pendent experiments normalized in this manner typicallyresulted in less than 10% variation in translation productsynthesized. Translation reactions were performed in retic-ulocyte lysate and wheat germ extract as described previ-ously (1, 41). Unless specified, reaction mixtures wereincubated for 1 h at 24°C, at which time translation wasterminated by freezing.

Aliquots of either 0.5 or 1.0 ,ul of the total translationproducts were added to sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) loading buffer,boiled for 5 min, and separated on 16% polyacrylamide gelsfor the standard Laemmli discontinuous buffer system (31)or 10% polyacrylamide gels for the Tris-Tricine buffer sys-tem (42). The gels were fluorographed and exposed to film at-70°C for times typically ranging between 2 h (e.g., prepro-lactin with the UTK leader) and 24 h (e.g., c-Src). Longerexposures were required for accurate quantification of someof the bands in Fig. 1. In all cases, translational efficiencywas determined from an exposure of the film in which theoptical density of the band(s) of interest was within themeasured linear region of the film (data not shown), using adensitometer (LKB 2222-020 Ultroscan XL).

Rate of ini'tiation. To facilitate measurement of initiationrates, translation was defined as initiated only if it resulted inthe synthesis of a full-length protein. Therefore, all transla-tion reactions which terminated prematurely or initiated atan AUG other than the authentic protein start site were notincluded in our assay. Using this definition, it was possible tomeasure cumulative initiations for the different RNA speciesby removing aliquots of a translation reaction at timedintervals and adding them to tubes containing aurin tricar-boxylic acid and 7-methylguanosine as initiation inhibitors(final concentrations, 1.0 x 10-4 and 4.0 x 10-3 M, respec-tively). Once initiation was blocked, translation was allowedto continue at 24°C for 1 h to ensure that elongation andtermination events had completed. After separation of 0.5 to1.0 ,u of the total reaction mixture by SDS-PAGE, densi-tometry of the resulting autoradiograms permitted estima-tion of cumulative initiations. The rate of initiation duringany period was obtained from the slope of a straight line fitto the relevant subset of the data points with a linearregression program.

RESULTS

A composite leader enhances translation. When the endog-enous leader sequences were replaced with a compositeleader containing the Xenopus 3-globin UTR and a syntheticinitiation site, the translation of a variety of polypeptides inreticulocyte lysate was dramatically improved. Translationof hybrid RNA encoding hamster PrP (Fig. 1, lanes 1 and 2),human ATIII (lanes 3 and 4), herpesvirus Vmw65 (lanes 5and 6), and canine SRP receptor a subunit (lanes 7 and 8) allshowed significant increases over that obtained with thecognate leaders. The largest measurable increase was ob-served with human ATIII; in this case, the composite leaderincreased translation by almost 300 times (compare lanes 3and 4). The increase with Vmw65 was even larger; however,the low levels of translation observed with the cognateleader precluded accurate measurement. Similar resultswere obtained with a wheat germ extract (data not shown).To separate the contribution to increased translation due

to optimizing the initiation site from that due to replacing theUTR, a series of different leader sequences was constructed.The sequences of relevant regions of the plasmids encodingthe different leaders are shown in Fig. 2. The SP6 promotersequence and the translation initiation site are both indi-cated. The first 11 nucleotides downstream of the SP6promoter are identical in all of these plasmids; therefore, the5' ends of the transcribed RNAs should all be the same. Therationale for the construction of each of the leaders was asfollows. The S leader is typical of plasmids which result fromcloning a coding region directly into the polylinker of com-

VOL . 1 l, 1991


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

2658 FALCONE AND ANDREWS

-_ -SRM-VW65- ATIII

II -PrP

LeaderendogenousUTK

+ + + ++ + + +

1 2 3 4 5 5 7 3FIG. 1. Comparison of protein synthesized from RNA with

cognate and UTK leader sequences. RNA synthesized by transcrip-tion of plasmids in vitro was translated in reticulocyte lysate. Thesame amount of RNA was added to all reactions. Coding regions:hamster PrP (lanes 1 and 2), human ATIII (lanes 3 and 4), herpes-virus protein Vmw65 (lanes 5 and 6), and canine SRP receptor asubunit (SR; lanes 7 and 8). The leader sequence used is indicatedbelow each lane. The migration positions of the full-size proteins areindicated at the right.

mercial expression vectors such as the pGEM or pSP series.The extraneous cloning sites 5' to the translation initiationsite were deleted from the S leader to generate the SDleader. This deletion also removes an ATG codon from theUTR which is not in frame with the coding region and lies ina poor initiation context. The KD leader is similar to the SDleader except that the initiation site now conforms to theconsensus sequence at the -3 position as well as at positions-2 through +4. In the K leader, a small open reading frame(ORF), which fortuitously occurs in the polylinker of pSP72,was added to the UTR of the KD leader. The nucleotidesequence surrounding this upstream ATG (and the one in theS leader) deviates markedly from the consensus for transla-tion initiation and therefore was not expected to affecttranslation measurably (29). Nevertheless, the addition ofthis region alters the translational properties of some codingregions, as described below. The UTR leader contains the

Xenopus ,-globin UTR with the initiation site compromisedat the -3 position. Finally, the UTK leader combines theXenopus P-globin UTR with an optimized initiation site.The coding regions subjected to more detailed analysis

were chosen from those readily available on the basis ofpreliminary experiments which demonstrated that transla-tion of these sequences was not complicated by frequentincorrect initiations or premature terminations (data notshown and Fig. 3). Two of the coding regions used containedadditional coding region mutations. In one mutant, referredto here as SRD4, amino acids 2 to 26 were deleted from theSRP receptor a subunit, substantially reducing the internalinitiations observed when the full-length molecule was ex-pressed by using the compromised leaders (data not shownand Fig. 3). The other mutant coding region contained aninternal deletion to permit competition experiments to beperformed on molecules with very similar but distinguish-able coding regions. This molecule, termed sPt, was derivedfrom preprolactin by deleting from the plasmid the regionencoding amino acids 2 to 57 of the mature portion of themolecule as described previously (2). More than 20 aminoacid residues at the amino-terminal end (from the pre region)as well as the carboxyl three-fourths of sPt are identical topreprolactin. As shown below, the difference in molecularweight is sufficient to allow sPt and preprolactin to be easilyresolved by SDS-PAGE.To permit direct comparison of translational efficiency,

equal quantities of RNA were added to translation reactionmixtures. The amount of RNA synthesized in the transcrip-tion reaction was measured with a fluorometer as describedabove. Control experiments (not shown) indicated that thefluorometry assay was sensitive enough to measure changesin RNA concentration that did not result in detectablealterations in the amount of translation products obtained.Furthermore, as shown below, the assay was reproducibleenough to permit accurate normalization of RNA synthe-sized from the same template in separate experiments.When the various coding regions were transcribed in vitro

and translated in either reticulocyte lysate (Fig. 3A) or wheatgerm extract (Fig. 3B), the translational efficiencies of the

CODnZ PEANS rood&Globin aft P_prbn PrPS

+ + + + s

+ + SD

+ + + + + + MD

A1TFAGGTGAC$CTATMMTACAAGC1TG.

AU I A

AT1TMGTGAC.CTATDOAATPCMOCTGA1CTD.ATGGwes -RMO

+ + + K

+ + + + + + umr

+ + + + + + UAK

ArrrMsTacrATb _ATbcAjacrr_AuIsA,u_mu,sil G, TE _s mPi

WH nn ~~~~CCIANo=

*S PSOMOTOS NZ WIOPU u-n NCUNCOI

FIG. 2. Plasmid sequences used to transcribe the leaders employed. The coding regions attached to each leader are indicated at the left,and the sequence is printed to the right of the leader name. The SP6 promoter is marked with the thin line below the sequence. The initiationsite is indicated with the thick underline. Relevant restriction sites in the plasmids are indicated. The upstream ATG and small ORF in theS and K leaders, respectively, are identified with lines above the sequences.

MOL. CELL. BIOL.

m ln Am --ut"

ucal

s RemT


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


A

B

I

4A 4f

._

Leader a * a1 n1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20 21 22 23 24 25 2627

Codingsequence Globin sPt Preprolactin PrP cSRC SRD4

FIG. 3. Translation of coding regions with the different leader sequences in reticulocyte lysate (A) and wheat germ (B). Fluorograms ofthe resulting polypeptides labeled with [35S]methionine and separated by SDS-PAGE are shown. Coding regions and leader sequences (asdefined in Fig. 2; * indicates cognate leader) are indicated below the fluorograms. For each coding region, equal amounts of the differentleader-bearing RNAs were translated. The photographs were aligned to approximate the relative migration positions of the differentpolypeptides with reference to molecular weight markers in each gel.

different constructs with identical coding regions fused to thedefined leaders varied by as much as 20-fold, as judged bytotal protein synthesized in 1 h. In all cases, the UTK leaderwith the favorable UTR joined to the optimized initiationsequence resulted in the largest amount of protein synthe-sized (compare plasmids designated UTK with the others ineach set). As expected, the lowest synthesis for most of theconstructs analyzed was obtained with either the S or SDleader, both of which have a C instead of an A at the -3position of the initiation sequence. The average increase insynthesis obtained when the S or SD leader was replaced bythe UTK leader was 9.8 + 5.3 (standard deviation)-fold. Asexpected, altering the initiation site such that it more closelyresembled the consensus for initiation by replacing the C inthe -3 position with an A increased the amount offull-lengthprotein synthesized (compare S and SD leaders with K andKD leaders in Fig. 3). This result is in good agreement withnumerous other studies on the effect of initiation site onsynthesis (reviewed in reference 29).

In many cases, the alteration in protein synthesis due tochanging the 5' UTR is quite dramatic (for examples, in Fig.3A, compare lanes 1 and 2 and lanes 11 and 12; in Fig. 3B,compare lanes 1 and 2, 7 and 8, 12 and 13, 16 and 17, 21 and22). For other RNAs, the variations are more subtle; forexample, lanes 12 and 13 in Fig. 3A illustrate a 23% increasein synthesis when the KD leader is replaced with the UTKleader. Although this value represents a relatively smallincrease, it is exquisitely reproducible. The average increasein synthesis measured in three independent experiments forthis molecule in reticulocyte lysate was 23.04% ± 6.48%(standard deviation). Similar relatively small increases insynthesis observed for the PrP coding region not only werereproducible but could be measured throughout the 60-minsynthesis reaction (see Fig. 5).Comparing the amount of synthesis obtained with the K

and KD leaders indicates that introducing a small ORF in theleader sequence can lead to unpredictable results. Globin

synthesis was not affected by the ORF (compare lanes 2 and3 in Fig. 3A and B). Moreover, deleting the ORF onlymarginally improved c-Src synthesis in reticulocyte lysateand had no effect in wheat germ extract (Fig. 3A and B, lanes20 and 21). However, the same alteration in leader sequenceresulted in increased synthesis of preprolactin in reticulocytelysate (Fig. 3A, lanes 11 and 12) but decreased synthesis inwheat germ extract (Fig. 3B, lanes 11 and 12). Although themore than 50% decrease in synthesis observed when theORF is removed from the UTR of preprolactin is veryreproducible (data not shown), it is difficult to interpret.When the synthesis of preprolactin in wheat germ extract iscompared for the S and KD leaders, a similar 50% decreaseis observed, suggesting that something in the KD leaderreduces synthesis in vitro.

Perhaps the most surprising result was the translation ofthe different coding regions with use of the UTR leader.Although this leader has a compromised initiation site,synthesis was in general better than for the leaders in whichthe UTR was derived from the plasmid polylinker regions (S,SD, K, and KD) even if those leaders contained an idealinitiation site (K and KD). In reticulocyte lysate, the in-crease in translation due to the UTR is particularly apparentfor c-Src and SRD4. In wheat germ extract, all of the codingregions except globin exhibit greater synthesis with the UTRleader than with either the K or KD leader.

Although maximum synthesis was obtained for all of thecoding regions with the UTK leader (the Xenopus P-globinUTR and an optimized initiation site), the amount of synthe-sis was also influenced by the coding region. Direct compar-ison of the different coding regions was possible after syn-thesis of the molecules in parallel reactions. Afterdensitometry of the autoradiograms, relative synthesis wascalculated by correcting for the number of methionines ineach molecule. The relative translational efficiency in retic-ulocyte lysate of each of the coding regions fused to theUTK leader was as follows: preprolactin, 100%; globin,

VOL . 1l, 1991

I

n

_ Ny CC _ crv1 cc v1 tr


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


z('2

8

6

4

2

0 25 50 75 100 0 25 50 75 100 0 20 40 60 80 I60% TRANSCRIPT % TRANSCRIPT % (UTK) sPt TRANSCRIPT

FIG. 4. Synthesis of the preprolactin and sPt coding regions in reticulocyte lysate at different RNA concentrations. Leader sequences: K,*, UTK; 0, 0, K; V, V, S. (A) Preprolactin transcript diluted with transcription buffer; (B) preprolactin transcript diluted with transcriptfor sPt with UTK leader (open symbols) and UTR leader (solid symbols); (C) preprolactin transcripts diluted serially with sPt transcript withthe UTK leader and translated in reticulocyte lysate. Maximum synthesis for S, K, and UTK leaders was fixed as 10 on the relative scaleshown. Error bars indicate the range in variation observed for the K leader in triplicate experiments. Tick marks indicate 2, 4, 6, and 8arbitrary units in each panel.

47%; sPt, 45%; PrP, 29%; c-Src, 5.8%; and full-length SRPreceptor a subunit, 2.8%. The amount of synthesis did notcorrelate with the length of the coding region, suggesting thatthe specific sequence rather than the size of the messagecontributes to the translational efficiencies measured here.However, since translational efficiency is measured as anaccumulation of full-length polypeptide, these differencesmay reflect alterations in rates of translation elongation (dueto pause sites, specific tRNA availability, etc.) or termina-tion. Moreover, the 3' UTR is identical only for all RNAswith the same coding region. Therefore, regulation involvingthis region of the RNAs may in part explain the observeddifferences in protein synthesis. This possibility appears lesslikely, since the 3' noncoding region has been reported tohave only a small effect on either RNA stability or transla-tion in cell-free systems (30).

Increased translation is not related to RNA concentration.Increases in translation similar to those reported here havealso been observed with use of chimeric mRNA in which theleader sequence of a highly expressed viral protein was fusedto different coding regions (11, 12, 17, 18). In these studies,increases in translation have been suggested to result fromincreased binding of a limiting factor by the chimeric RNA,since differences in translational efficiency were observedonly at high mRNA concentrations (18). To test this possi-bility for the UTK leader, translation was examined atvarious RNA concentrations and in direct competition ex-periments.

If the amount of a specific RNA is reduced in the reticu-locyte lysate translation reaction, the amount of proteinsynthesized is also reduced, as expected. However, at anyparticular preprolactin RNA concentration, the trend oftranslational efficiency for the different leader sequencesobserved in Fig. 3 is unchanged (Fig. 4A). This trend is notconsistent, with the difference in synthesis being due tobinding of a limiting factor. Similar results were obtained fortranslation of other coding regions with these leader se-quences (PrP in reticulocyte lysate as well as preprolactinand globin in wheat germ extract; data not shown). In Fig.4B, the preprolactin RNA concentration was reduced byserial dilution with transcript for sPt. By dilution with an

alternate transcript, the total RNA concentration in thereactions was held constant. The results of experimentsusing sPt with the UTR leader and with the UTK leader asdiluent are shown in Fig. 4B. The pattern of synthesis shownis essentially the same as in Fig. 4A; therefore, the increasein translational efficiency due to the UTK leader is essen-tially independent of template RNA concentration. Theexperiment shown in Fig. 4B can also be used to assaycompetition for translation factors directly. If the differenttemplates compete equally for translation factors, then pre-prolactin synthesis as mediated by any of the leaders willdecrease linearly as this transcript is replaced with one fusedto the UTK leader encoding sPt. To maximize the spread inthe data, the highest preprolactin synthesis was set to 10arbitrary units for each leader sequence. When plotted inthis fashion, unequal competition ofUTK leader-bearing sPtRNA with any of the preprolactin RNAs would be expectedto result in a deviation from the line plotted in Fig. 4C.Synthesis of preprolactin with each of the leaders decreaseslinearly, independent of overall translational efficiency, con-firming that significant competition for a required factor isnot observed.

Translational efficiency correlates with rate of initiation.Translational initiation events which resulted in the synthe-sis of full-length hamster PrP were measured at various timepoints for RNAs with the different leader sequences. Thecumulative number of initiations at any time point wasgreater for RNA with the UTK leader than with the UTR,KD, or endogenous PrP leader sequence (Fig. 5). Twomechanisms could contribute to a larger number of cumula-tive initiations. First, the RNA bearing the UTK leadermight initiate more quickly when the synthesis reaction isfirst started; i.e., the lag time before full-length molecules areobserved may be reduced. Second, initiation may take placeat a higher rate over the entire reaction period with the UTKleader. To assess the relative importance of each of thesemechanisms, initiation rates were determined from theslopes of the linear regression lines (Fig. 5B) fit to the dataobtained for the first 20 min of translation. During this timeperiod, the rate of initiation clearly varies for the differentleader sequences. These regression lines also indicate that

MOL. CELL. BIOL.


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


10

z~~~~~~~~~~~~~~~~~0

iEo 06 .4 4~~~~~~~~~~~~~~~~~~

2 2

v~~~~~~~~~~

0 0 *0 10 20 30 40 50 60 0 5 10 15 20 0 5 10 15 20 25

TME (MIN) TIME ()MN) INITIUATON RATE

FIG. 5. Evidence that initiation rate determines the relative level of PrP synthesis with the different leader sequences. (A) Cumulativeinitiations resulting in synthesis of full-length PrP during 60 min of translation in reticulocyte lysate. (B) Cumulative initiations during the first20 min of synthesis presented on an expanded scale to illustrate individual data points and regression lines. Initiation rate for each leader wasmeasured from the slope of the regression lines. (C) Total synthesis of PrP with each of the different leaders after 60 min of translation,correlating with the initiation rate measured in panel B. Leader symbols: *, UTK; E, UTR, *, KD; V, cognate.

there was not a significant difference in the observed lagtimes before initiation occurs (Fig. 5B). Moreover, thedifferences in initiation rates measured during the first 20 mincorrelate with differences in total protein synthesized (Fig.5C). Therefore, it appears that the increase in proteinsynthesized from RNA bearing an optimized leader resultsfrom an increase in the number of productive initiationevents occurring during the synthesis reaction.Taken together, the results presented in Fig. 4 and 5

suggest that the increase in translation observed with theoptimized leader results from increased utilization of trans-lation factors rather than from increased affinity for a limitingfactor in the lysates. However, since we are measuring theproducts of the translation reaction and not analyzing initi-ation directly, it is possible that the differences observed aredue to a mechanism which selectively abolishes the transla-tion of specific RNAs. Two of the easily tested possibilitiesare rapid degradation or specific inactivation of the lessefficiently translated RNAs.To test these suppositions, translational efficiency was

measured for different leader sequences in staged translationreactions. In these experiments, reaction mixtures wereassembled on ice without the added energy components(ATP, GTP, creatine phosphate, creatine kinase, and aminoacids) and the reticulocyte lysate was depleted of smallmolecules by salt exchange on Sephadex G-25. The reactionmixtures were placed at 24°C to start translation, and thepremixed energy components were added after 0, 15, or 30min. For all of the reactions, translation was allowed tocontinue for 60 min after the energy components were addedand then terminated by freezing. The results obtained withPrP and c-Src coding regions are shown in Fig. 6. Asexpected, the relative translational efficiency of the differentleader sequences follows the same pattern seen above at anyof the time points assayed (compare the amount of proteinsynthesized in lanes 1 to 3, 4 to 6, and 7 to 9 for each codingregion). However, the amount of synthesis obtained withany particular leader decreases with increasing incubationtime before the addition of the energy components (comparelane 1 with lanes 4 and 7). These results are not consistentwith either selective RNA inactivation or degradation as anexplanation for the differences in translational efficiency.

The results obtained are consistent with a continuous declinein the availability of a required translation component.Leader sequence structure. Leader sequence secondary

structure and length have both been proposed as determi-nants of translational efficiency (14, 18, 19, 22, 27, 46).

- 94-- 67- 43

PrP

Leader ETime 0

30

2014

N U C h U 1 N U0 0 15 15 15 30 30 30

cSRC

LeaderTime

S K U K USKU0 0 0 15 15 15 30 30 30

946743

30

20

14

1 2 3 45 6 7 8 9FIG. 6. Stability of in vitro transcription products in reticulocyte

lysates. Translation of RNAs encoding PrP (A) and c-Src (B) withthe different leader sequences was initiated by adding energycomponents and amino acids after 0, 15, and 30 min of incubation at24°C (lanes 1 to 3, 4 to 6, and 7 to 9, respectively). Leader sequencesare indicated below the lanes. Positions of migration are indicated inkilodaltons on the right.

VOL . 1 l, 1991


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


Specifically, it has been suggested that lack of stable sec-ondary structures, such as hairpin loops, in the UTR con-tributes to the translational efficiency mediated by highlyefficient leader sequences (18). For this reason, we examinedthe leaders used here for evidence that secondary structurecontributes to differences in translational efficiency. Stablesecondary structures (those with a AG of ' -30) are notpredicted for any of the leaders illustrated in Fig. 2, since thecalculated AG values did not exceed -5. Values for G werecalculated only for the leader sequences and did not includethe coding regions because such calculations are of limitedvalue over extended regions. Furthermore, regions of RNAduplex structures within the coding region of an mRNA havebeen shown not to block translation elongation and aretherefore unlikely to be relevant to our measurements (33).

In addition to these empirical measures, we examined therole of RNA structure in translational efficiency directly.Heat denaturation, magnesium titrations, and linearizationof the plasmids before transcription were used alone and incombination to alter RNA structure before translation. Todenature RNA, transcript was heated to 70°C for 5 min andthen chilled rapidly on ice. This procedure should abolishlonger-range secondary structures but will have limitedeffect on short-range structures. When relative translationwas measured for the heat-denatured RNAs, the order andmagnitude of translational efficiency mediated by the dif-ferent leaders were unchanged (data not shown). Similarly,altering the concentration of magnesium in the reactionmixtures (from 2.0 to 3.6 mM in 0.4 mM steps) affectedoverall translational efficiency as expected (8, 15, 32) but didnot change the relative efficiency of the different leaders(data not shown). Finally, when plasmids were linearizedbefore transcription either alone or in combination with heatdenaturation, a small increase in translation was observedfor all of the leaders (data not shown).

DISCUSSION

In an effort to exploit the Xenopus ,-globin leader se-quence to enhance the efficiency of translation in vitro, thecoding regions of a number of different genes were attachedto it via a synthetic translation initiation sequence. Transla-tion mediated via this leader sequence, termed UTK, was10-fold to greater than 300-fold higher than that obtainedwith the endogenous leader sequence (Fig. 1). However,since the natural mRNAs for these molecules have not beensequenced, we cannot be certain that the cognate leaders arecomplete. Therefore, while these results accurately reflectthe translational efficiency obtained in practice, they maynot indicate the true synthetic capacity of the endogenousmRNAs. Nevertheless, translation was approximately 10times higher for the same coding regions with the UTKleader than when expressed in standard in vitro expressionvectors without an optimized leader (Fig. 3). This samemagnitude of increase was observed when the tobaccomosaic virus leader sequence was added to a chloramphen-icol acetyltransferase gene and the chimera was expressed inXenopus oocytes (13). Similar magnitudes of translationalenhancement have been reported for a variety of viralleaders in Escherichia coli (11) and chimeric mRNAs con-sisting of the tobacco mosaic virus leader or the alfalfamosaic virus RNA4 leader sequence fused to barley a-amy-lase expressed in wheat germ extract (17, 18). However, theincreases in translational efficiency reported here are largerthan those reported for the alfalfa mosaic virus RNA4 leadersequence fused to interleukin-l, or the rabbit a-globin

leader fused to barley a-amylase, both expressed in wheatgerm extract (18).For the different leader sequences examined here, the

increase in synthesis obtained due to the addition of anoptimized initiation site was roughly similar to that obtainedby exchanging the UTR for that from Xenopus ,B-globin.However, the two sequences together outperformed eithersequence alone for all of the coding regions tested (Fig. 2).The effect of the two sequences was more than additive forthe expression of globin and c-Src in both reticulocyte lysateand wheat germ extract and for sPt in wheat germ extract.These results strongly suggest that both sequences contrib-ute to the efficient initiation of translation in vitro.The fidelity of reticulocyte lysate and wheat germ extracts

for assaying the translation of SP6 transcription products hasbeen carefully characterized (9, 28, 35). In some experi-ments, the fidelity of initiation of translation by cell-freesystems has been shown to vary with the ionic conditions ofthe reaction (8, 15, 28). However, the effects of leadersequence on translation reported here are qualitatively thesame over a wide range of conditions (e.g., magnesiumconcentrations from 2.0 to 3.6 mM). Other experiments haveillustrated directly the ability of wheat germ and reticulocytelysate systems to mimic whole-cell assays (9, 35). Therefore,we are confident that the results shown here are not due todifferences in ionic conditions or RNA stability in thecell-free systems. Moreover, our own control experiments(Fig. 6 and others not shown) suggest the RNA used as atranslation template not only is stable but remains transla-tion competent throughout the reactions. In one such con-trol, when translation was allowed to proceed for 1 h andthen supplemented with labeled amino acid, as well asadditional ATP and lysate, the same pattern of translationalefficiency was observed for the different coding region-leader combinations (data not shown). These results confirmthat differences in observed translational efficiencies are notdue to the relative stability of the RNA in vitro. Further-more, the differences in translation are not a function of thetranslation system, since the same general trends are ob-served in both reticulocyte lysates and wheat germ extracts.The data presented in Fig. 4 to 6 strongly suggest that the

increase in translation observed with the UTK leader is dueto an increase in the rate of initiation. Our results are notconsistent with an earlier proposal suggesting that increasedtranslational efficiency mediated by viral leader sequencesresults from increased affinity of the leader sequence for oneor more limiting factors in the translation reaction mixture(18). Similarly, our results are not consistent with an alter-native proposal that the increase in efficiency results from adiminished requirement for a limiting factor (18). One pos-sible explanation consistent with our data would be reducedrequirement of the UTK leader for the unwinding activity ofthe cap-binding complex (18). At present it is not clearwhether the differences that we observe between the behav-ior of the UTK leader and that observed for viral leadersrepresents a real difference in the mechanism(s) responsiblefor enhancing translation, since direct comparisons have notbeen reported.There were several differences in the relative translational

efficiency mediated by the compromised leader sequencesthat would not have been predicted by the modified scanningmodel for translation. The most intriguing of these is theability of an efficient untranslated region to more thancompensate for a compromised initiation sequence. Thiseffect is illustrated by the relative amount of protein synthe-sis obtained with the KD and UTR leaders (Fig. 3 and 5). In

MOL. CELL. BIOL.


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


contrast to the tenets of the scanning model, translation canbe more efficient with a leader sequence compromised by anA-to-C substitution at the critical -3 position than with anunrelated leader containing an optimal initiation sequence.For example, translation of sPt, preprolactin, PrP, c-Src,and SRD4 with the UTR leader (initiation sequenceCCCAUGG) is equal to or greater than for the same codingregions with the KD leader (initiation sequence ACCAUGG). Moreover, even in the identical context of the SDand KD leaders, translation was not always improved by theoptimized initiation sequence (e.g., c-Src). Although theseresults are at variance with some of the predictions attrib-uted to the scanning model, they are not inconsistent with ascanning mechanism per se. Furthermore, optimizing theinitiation sequence within the UTR leader improved thetranslation of most of the coding regions tested. Together,these results argue that the leader sequence UTR andinitiation site interact to promote efficient translation invitro.Another surprising finding was the effect of introducing a

small ORF into the leader sequence. This modification wasexpected to promote leaky scanning and thereby reduce theefficiency of translation of the various coding sequencestested (29). Instead the results obtained were unpredictable,with the ORF having no measurable effect on the translationof either globin or c-Src and opposing effects for translationof preprolactin in reticulocyte lysate and wheat germ ex-tract.

Several other more subtle differences were observed be-tween the wheat germ extract and reticulocyte lysate sys-tems (Fig. 3). To some extent these alterations may reflectthe differences reported previously for selection of AUGcodons in plants and animals (35). However, in contrast toresults showing that translation was always insensitive to the-3 position, we find that translation initiated by the ,B-globinUTR is improved by a C-to-A substitution at this site.

It is often difficult to extend the results obtained withcell-free assays to in vivo systems. However, high-efficiencytranslation has been observed for the Xenopus ,B-globinleader in Xenopus oocytes (lOa, 30). In addition, there issome evidence suggesting that the relative efficiencies ofother leader sequences in vitro are similar to those observedin vivo (11-13). Nevertheless, other regulatory systems notreproduced in vitro are likely to regulate translational effi-ciency in whole cells. For example, many recent experi-ments suggest that translation may also be regulated bysequences in the RNA 3' of the coding region (reviewed inreference 16). However, this region has been reported tohave little effect on RNA stability or translation in cell-freesystems (30). In addition to the UTRs, the coding region mayalso influence translation, and experiments addressing thiseffect are in progress. Eventually a whole-cell model willhave to be devised in which it is possible to address therelative importance of the sequences throughout the entiremRNA in mediating efficient translation in vivo.

ACKNOWLEDGMENTS

Research funds were provided by a grant from the MedicalResearch Council of Canada. D.W.A. was the recipient of an MRCof Canada scholarship.

REFERENCES1. Andrews, D. W. 1989. Examining protein translocation in cell-

free systems and microinjected Xenopus oocytes. Biotech-niques 7:960-967.

2. Andrews, D. W., E. Perara, C. Lesser and V. R. Lingappa. 1988.

Sequences beyond the cleavage site influence signal peptidefunction. J. Biol. Chem. 263:15791-15798.

3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Currentprotocols in molecular biology. John Wiley & Sons, New York.

4. Banerjee, A. K. 1980. 5' terminal cap structure in eucaryoticmessenger ribonucleic acids. Microbiol. Rev. 44:175-205.

5. Berkner, K. L., B. S. Schaffhausen, T. M. Roberts, and P. A.Sharp. 1987. Abundant expression of polyomavirus middle Tantigen and dihydrofolate reductase in an adenovirus recombi-nant. J. Virol. 61:1213-1220.

6. Brown, P. H., S. Daniels-McQueen, W. E. Walden, M. M.Patino, L. Gaffield, D. Bielser, and R. E. Thatch. 1989. Require-ments for the translational repression of ferritin transcripts inwheat germ extracts by a 90-kDa protein from rabbit liver. J.Biol. Chem. 264:13383-13386.

7. Cigan, A. M., and T. F. Donahue. 1987. Sequence and structuralfeatures associated with translational initiator regions inyeast-a review. Gene 59:1-18.

8. Daniels-McQueen, S., D. McWilliams, S. Birken, R. Canfield, T.Landefeld, and I. Boime. 1978. Identification of mRNAs encod-ing the a and P subunits of human choriogonadotropin. J. Biol.Chem. 253:7109-7114.

9. Dasso, M. C., and R. J. Jackson. 1989. On the fidelity of mRNAtranslation in the nuclease-treated rabbit reticulocyte lysatesystem. Nucleic Acids Res. 17:3129-3144.

10. Elroy-Stein, O., T. R. Fuerst, and B. Moss. 1989. Cap-indepen-dent translation of mRNA conferred by encephalomyocarditisvirus 5' sequence improves the performance of the vacciniavirus/bacteriophage T7 hybrid expression system. Proc. Natl.Acad. Sci. USA 86:6126-6130.

10a.Falcone, M., and D. W. Andrews. Unpublished data.11. Gallie, D. R., D. E. Sleat, J. W. Watts, P. C. Turner, and

T. M. A. Wilson. 1987. The 5'-leader sequence of tobaccomosaic virus RNA enhances the expression of foreign genetranscripts in vitro and in vivo. Nucleic Acids Res. 15:3257-3272.

12. Gallie, D. R., D. E. Sleat, J. W. Watts, P. C. Turner, andT. M. A. Wilson. 1987. A comparison of eukaryotic viral 5'leader sequences as enhancers of mRNA expression in vivo.Nucleic Acids Res. 15:8692-8711.

13. Gailie, D. R., D. E. Sleat, J. W. Watts, P. C. Turner, andT. M. A. Wilson. 1988. Mutational analysis of the tobaccomosaic virus 5'-leader for altered ability to enhance translation.Nucleic Acids Res. 16:883-893.

14. Gehrke, L., P. E. Auron, G. J. Quigley, A. Rich, and N.Sonenberg. 1983. 5'-Conformation of capped alfalfa mosaicvirus ribonucleic acid 4 may reflect its independence of the capstructure of cap-binding protein for efficient translation. Bio-chemistry 22:5157-5164.

15. Gerstenfeld, L., J. C. Beldekas, C. Franzblau, and G. E.Sonenshein. 1983. Cell-free translation of calf type III collagen.J. Biol. Chem. 258:12058-12063.

16. Jackson, R. J., and N. Standart. 1990. Do the poly(A) tail and 3'untranslated region control mRNA translation? Cell 62:15-24.

17. Jobling, S. A., C. M. Cuthbert, S. G. Rogers, R. T. Fraley, andL. Gehrke. 1988. In vitro transcription and translational effi-ciency of chimeric SP6 messenger RNAs devoid of 5' vectornucleotides. Nucleic Acids Res. 16:4483-4498.

18. Jobling, S. A., and L. Gehrke. 1987. Enhanced translation ofchimeric messenger RNAs containing a plant virl untranslatedleader sequence. Nature (London) 325:622-625.

19. Johansen, H., D. Schuimperli, and M. Rosenberg. 1984. Affectinggene expression by altering the length and sequence of the 5'leader. Proc. Natl. Acad. Sci. USA 81:7698-7702.

20. Klausner, R. D., and J. B. Harford. 1989. Cis-trans models forpost-transcriptional gene regulation. Science 246:870-872.

21. Klemenz, R., D. Hultmark, and W. J. Gehring. 1985. Selectivetranslation of heat shock mRNA in drosophila melanogasterdepends on sequence information in the leader. EMBO J.4:2053-2060.

22. Konarska, M., W. Filipowicz, H. Domdey, and H. J. Gross. 1981.Binding of ribosomes to linear and circular forms of the 5'

VOL. 11, 1991


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


terminal leader fragment of tobacco-mosaic-virus RNA. Eur. J.Biochem. 114:221-227.

23. Kozak, M. 1978. How do eucaryotic ribosomes select initiationregions in messenger RNA? Cell 15:1109-1123.

24. Kozak, M. 1986. Point mutations define a sequence flanking theAUG initiator codon that modulates translation by eukaryoticribosomes. Cell 44:283-292.

25. Kozak, M. 1987. An analysis of S'-noncoding sequences from699 vertebrate mRNAs. Nucleic Acids Res. 15:8125-8148.

26. Kozak, M. 1988. A profusion of controls. J. Cell Biol. 107:1-7.27. Kozak, M. 1988. Leader length and secondary structure modu-

late mRNA function under conditions of stress. Mol. Cell Biol.8:2737-2744.

28. Kozak, M. 1989. Context effects and inefficient initiation atnon-AUG codons in eucaryotic cell-free translation systems.Mol. Cell. Biol. 9:5073-5080.

29. Kozak, M. 1989. The scanning model for translation: an update.J. Cell Biol. 108:229-241.

30. Krieg, P. A., and D. A. Melton. 1984. Functional messengerRNAs are produced by SP6 in vitro transcription of clonedcDNAs. Nucleic Acids Res. 12:7057-7070.

31. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

32. Lightfoot, D. A. 1988. Magnesium-dependence of in vitro trans-lation programmed by gene-specific mRNAs. Nucleic AcidsRes. 16:4164.

33. Lingelbach, K., and B. Dobberstein. 1988. An extended RNA/RNA duplex structure within the coding region of mRNA doesnot block translational elongation. Nucleic Acids Res. 16:3405-3414.

34. Lodish, H. F. 1976. Translational control of protein synthesis.Annu. Rev. Biochem. 45:39-72.

35. Lutcke, H. A., K. C. Chow, F. S. Mickel, K. A. Moss, H. F.Kern, and G. A. Scheele. 1987. Selection of AUG initiationcodons differs in plants and animals. EMBO J. 6:43-48.

36. McGarry, T. J., and S. Lindquist. 1985. The preferential trans-

lation of drosophila hsp70 mRNA requires sequences in theuntranslated leader. Cell 42:903-911.

37. Mierendorf, R., and J. Jendrisak. 1985. Optimizing transcriptionperformance of riboprobe SP6 and T7 RNA polymerases.Promega Notes 1:2-6.

38. Morle, F., B. Lopez, T. Henni, and J. Godet. 1985. a-Thalas-saemia associated the deletion of two nucleotides at position -2and -3 preceding the AUG codon. EMBO J. 4:1245-1250.

39. Perara, E., and V. R. Lingappa. 1985. A former amino terminalsignal sequence engineered to an internal location directs trans-location of both flanking protein domains. J. Cell Biol. 101:2292-2301.

40. Ray, B. K., T. G. Lawson, J. C. Kramer, M. H. Cladaras, J. A.Grifo, R. D. Abramson, W. C. Merrick, and R. E. Thach. 1985.ATP-dependent unwinding of messenger RNA structure byeukaryotic initiation factors. J. Biol. Chem. 260:7651-7658.

41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

42. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1-100 kDa. Anal. Biochem. 166:368-379.

43. Sonenberg, N. 1981. ATP/Mg+ +-dependent cross-linking of capbinding proteins to the 5' end of eukaryotic mRNA. NucleicAcids Res. 9:1643-1656.

44. Tahara, S. M., M. A. Morgan, and A. J. Shatkin. 1981. Cyclicnucleotide-independent protein kinases from rabbit reticulo-cytes. Identification and characterization of a protein kinaseactivated by proteolysis. J. Biol. Chem. 256:7691-7694.

45. Walden, W. E., M. M. Patino, and L. Gaffield. 1989. Purificationof a specific repressor of ferritin mRNA translation from rabbitliver. J. Biol. Chem. 264:13765-13769.

46. Weyer, U., and R. D. Possee. 1988. Functional analysis of theplO gene 5' leader sequence of the Autographa californicanuclear polyhedrosis virus. Nucleic Acids Res. 16:3635-3653.

MOL. CELL. BIOL.


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

start site contribute to efficient initiation of translation in vitro

Documents