.J. Mol. Rid. (1992) 226. 609.-621

Translation Inhibition by an mRNA Coding Region Secondary Structure is Determined by Its Proximity to the AUG Initiation Codon Stephen A. Liebhabert, Howard

Faith Cash and Susan S. EshlemanS

Hughes Medical Institute, Departments of Emetics mad Alediciw C’niversity of Pennsylvania School of Medicine Philadelphia PL4 lRlOMlJ.i, l’.S.d

In the present study we investigat’e the impacat of highly stable coding region secyjndary strucatures on tnRSA t’ranslation efficiency. Hy int,roducing antisense segments into t,hr ~3’ tlon~trullslatrtd region of human a-globin tnRX\;A we are able to synthesize a series of’ transcripts in which site-specific secondary structures are introduced without altering the primary structure of the 5’ non-translated region. the coding region. or the encoded protein I)roduct. (‘oding region duplexes in close proximity to the a initiation codon are fonncl to inhibit translation severely to a degree equal to t’hat of a duplex that extends into thri .?’ non-translated region. In contrast, mRK\;As containing duplexes positioned further 3 in the coding region translate at levels that’ are significant~ly higher although are still helow those of nativcx r-globin mRNA. The primary determinant of translation inhibition b-y cwfing region duplexes appears to be t’he proximity of t’he duplex to the =zu(: initiation ~~~tlon and reflects a parallel inhibition of monosome formation. These data demonstrate that extensive wding region secondary structures suppress translat~ion to a tninimal or to a subst.antial degree depending on their distancae frotn the initiation codon. Kr!yworrls:

traiista,tion:

sewndary

structure:

1. Introduction The t.ranslational eficienc~y of an mRNA is determined by its structure ant1 by it,s interactions with cellular factors (for revilws. see Moldave, 19%: London it 01.. 19%). The structural det’erminant,s of t~ranslational efficiency may be encoded in the pritnary sequen(ae of the tnRSA as well as in its secondary and/or higher order structure. The presence of a 5’ (ap (Muthukrishnan rt nl., 197.5: Sonenberg. 198%) and an appropriate consensus sequenw surrounding the initiation codon, AU(: (note that X is used in the text to refer to the initiation codon: Kozak. 1984, 1986a: Edery et al.. 1989). are &ar examples of primary structural det,erminants of translational efficiency. In prokaryotic mR?iAs. both local and long-range secondary- structures can also alter translational efficiency hy hkwking interactions of the 30 S ribo-

1 Present address: L)epart,nirnt of Patholog,v. IIniversity of Pennsylvania School of MedicGne. Philadelphia. P.5. I9lO-C. (‘A.\.

mRN.4:

gene expression;

gtobin

tnK,Ni\

somal subunit with the Shine-l)alparno sit.e and && (Iserentant & Fiers. 1980: Hall rt ~1.. 1982). Studies of inhibition of eukaryoticn mRN.A translation by mR8SA/cDNA (Patterson cut nl., 197i; Hastie & Held. 197X: Privalsky 8: Bishop. 1982; l,iebhaber et nl., 1984; Shakin & Liehhaber, 1986), mRNA/cRSA (Melton, 1985) or int,ramolecular duplexes (Kozak. 1980. 19X6h, 19X9: l’elletier & Sonenberg. 1985: Spena it al.. 1985: Galili it nl.. 1986: Parkin et al.. 1988; Edery rt crl.. 1989) suggest that the relationship of secondary struc>ture to mRSA translation is complex. reflecting a number of variables. These variables include the size. position and stability of secondary st,ructurrs (references as above), as well as the cellular environment (Kozak. 19X8, 1989), and potential destabilization of duplexes by a variety of translat.ionallv relevant melting activities acting at the levels of iriitiation ot elongation (Liebhaber et al.. 1984: Ray ef nl.. 19X5: Shak’in & Liebhaber, 1986; Rozen rt rtl.. 19HO: for a revifw. see Spena et al., 1985). \\‘hile the above cited studies demonst.rate that translation is commonly inhibited by duplexes involving ACG and extending into t,he 5’ ntrntranslatrtl region. the effect’s of secwndary st ruc~t~ures

(ii0 situated wit,hin the cwhng rrgions of’ vukaryotit~ friR,NAs arc lrss well understood. St~utliw show that t hr hybridization of’ mRSA to (~J)K’x (Liebhabt~r rl ~1.. 1984: Shakin & Liebhaber. 19%) or c*RNA (!Uelton. 1985) fragments within the caoding region may have little or no effect on t,ranslation. However, such intermolecular duplexes may not accurat,rlJ rrflrcst t hr. irnf~wt of’ intramolec*nlar src~~rdq structures on translation. For rlxamplr. the demor~ stration of discaontinuous t~ranslatJional elongat~ion >Llollg a number of native mRiK;r\s has led to the spe(~ulation t,hat intramolecular duplexes may in facat block the progress of the 80 S ribosome (T’rotzrl k Morris. 1974; Smith. 1975: 1,izardi d al.. I!)$!): (‘andelas uf al.. 19%). A s\-st,ematic invrstigat’ion of thr relationship l&wren int~rarnolrc~ular c*odinp region duplexes in mRSAs and translation efTic+wcy is prwcwtly lacking. In the present study wt. begin to charact)erize the impact of rnRSSA coding region srcsondary strut.t ures on translation. The data demonstrate t’ha,t the rfiwts of such structures on overall t~ranslatJional rfFi&n~~y are critically dependent on their tbxact position relativr t)o the initiation cotion.

2. Materials and Methods

All recombinant manipulations were carried out using standard protocols (Maniat,is rt al.. 1982). Restric%ion and modification enzymes were purchased from eit,her Sew England Biolabs (SEBL. Beverly MA) or RorhringerMannhrim (HM. Tndianapolis 1%). and were used as suggested by the supplier. Linkers were purchased from NERT, and were phosphorylated before use. A full-length a-globin cl)NA. pSl’6ha2FL, extending from -37 (t)hr zAof AUG is defined as + 1) to an Al 8 tail was construct,ed by joining, at a common Xc01 site (+ 1). an a-globin cDN’A fragment, containing the full-length -5’ non-translat,ed rrgion to an r-globin (&DNA fragment containing the normal coding and full length 3’ nontranslated region. These fragment,s were isolated from 2 previously reported VDNA a-globin plasmids. pSP64cDKAha2 (MorlC d al., 1986) and pSa (Shakin Kr T,iebhaber. 1986). respectively. The a-globin transcript synthesized frorn the pSP6ha2FL plastnid contains a fulltrngt,h a-globin mRNA with an additional 27 bases in the 5 non-translat~ed region upst’rram from t’hr r-globin mRNA seyuences; 23 bases are derived frorn the vect,or polylinker and 4 are from the 5’ flanking region of the a-globin gene (Morlb ut al., 1986: see Fig. I ). (ii)

(~omtmction

of’ a-ylobin

c1)XA.s

with

antisenw

inserts

(‘onstruc%ions of transcription vectors rncoding a-globin mRNAs wit,h defined double-stranded regions were carried out as diagrammed in Fig. I. The fulllength a-globin cl)NA (pSP6haBFL) was linearized at a unique Apa, site in the 3’ non-translated region (+480) and cDNA fragments encoding various segments of the mRNA were ligated into this site. cc-Globin cDNAs with 5’ termini at -14. +I. +7. f18. +43, and +I53 were generated by Ha131 exonuclease digestion followed by

and gene-clrrivt~tl) k~asflsirl thtb 5’ Ilorl-translatt~tl r.c~gionof pST%hadFT, was caarried out t,,v oligonuc,lrotitlr-clirc,c,trtl site-sprcaific. nlut,agtknc+ (Zollrr & Smith. I!#:- Sphl SC01 digc&on and rxc*hangrtl with thf, c.or.I.~s~Jo~~(liI~~ 5’ Ilol;-t ranstated region of’ tht, qglobin antl tlvrivativf~ IX anti d:( c,l)N;\s (see atjove). Thr prfviic~tt~d df*letion Mas c~oniirmed b> dideoxy srquf~nc~ analysis (Sangrr rot rrl.. l!fii) [wing a primer (~orrrsponding to thfl seqwnw T,‘(:C’T.I( ‘AATTA4AL\TA(‘ATT.~I\(‘(‘3’ toc~atrtl .X to :I8 bases 5’ to thcx Sl’6 promot,f~r. ‘l’hc mutatfd f.I)SA inwrt prrdicts th(s synthtssis of iI full-lf~ngth a?-gtot)iIl rtiKXj:i irrtrritionatl~ rrtaining 2 basrs ((:A) at its 5’ tf.rmitius t’rotrl t Ilr vfWtor t.o ensure rflic.irnt Se6 tr;rIjsc.ription (Sollazr/.o cf trl 1988). (,1)X:\ vlonw in I)Sl’(il atlfl their ~~0rrf~sfJo~iflin~transcribed mRNAs \vith this shortf~nf~tl 5’ Irotl~tr;rtjalatPd region art’ wtbrrrd to with thr delta (A) pwti \ (iv) F//II-ten!& /I-,y/ohi?l c/),V.-I Sl’fi transcript,ion of a full-lrugth ,Cglobin (.lJNA in the pSP64 vec,tor (pSl’k/lc; ref :15. a generous git% of’ It. Spritz, t Abbreviat,ions used bl~, base-pair(s): RF. replic~ativf~ form; TAtT. t&on-a&-urea: u.v.. ultraviolet: nt. nuc~leotidr(s).

mRNA AUG

Bructure

UAA

BSSHII -14156 -14 1

Secondary

ApoLI

Ai01

Sal I

And Translation

611

Madison WI) results in a full-length /5globin mRh-8 which contains an additional 19 vector-derived bases 5’ to the native p-globin mRNA sequence and a 68 base poly(A) tail. (1~) Tn vit,ro tmnscriptiorc

-

18 43 57 85 108 153 851163 851349 2091366 2721368 I

I

/

..:“--i

,A? APO1

.&/I

4 *

(b)

AUG f

Prior to transcription, pSP6hcr2FL and each of its derivative plasmicls (Fig. 1) were linearized 3’ to the cDXA insert’ with SalI; pSPkpc was similarly linearized with HlndTTT. Each linearized plasmid was extracted with phenol/chloroform. precipitated with ethanol and dissolved in water at’ a concentration of 0.5 pg/pl. .4 total of 0.5 pg of each linearized plasmid was added to 50 {tl of transcription buffer mix. The 1 x tranwription mix 6 mu-MgCl,, contains 40 mw-Tris . HCI (pH 7.5). 2 mM-spermidine. 500 FM each of ATP. (‘TP and HTTP, 50 ~svGTP, 500 /civ-‘mG(li’)pppG. 0.1 ~1 [c~-~‘P](‘TI’ 100 Ci/mM. Amrrsham), anti (10 mCi/ml. 10 m>l-dithiothreitol. ,4 t,otal of 5 units of SP6 RNA polymerase (R’EBL) was added to the reaction mix and incubated for 60 min at 40°C. The reaction \vas trrmnated by placing it on ice and then desalted and depleted of unreacted nucleotides by passing it over a (SO yuickspin column (Borhringer-Mannhrim. Tndianapolis, TX) pre-washed with sterile distilled water. .I 2 ~1 portion of the excluded t,ranscription products w-as rlectrophoresed M-urea gel (18 \2. for 1 h), transon (’‘1 5”’ 0 acylatnide/8 ferred to n’hatman 3M paper. and exposed to Kodak XAIi-5 film in the presence of a (‘ronrx intensifying screc’n at - XO”(‘. The relative c*oncentration of each transcript was det,ermined from the band intensities on the gel autoradiographs by soft laser densitomrtry ((‘omput,ing Densitometer: Ylolecular Dynamics. Sunnyvale. (‘A). A4ll autjoradiograph signals were determined wlthin the linear range of densitometric detection,

I

\

“AAAl8 (c 1

Figure 1. (‘onstruction of transcription vectors that human mRXAs with site-specific encode cc-globin duplexes. (a) Segments of the a-globin cDNA duplicated in the 3’ non-translat)ed region. The 1st’ line shows a diagram of the full-lengt,h human &globin cDNA insert present in the pSP6 vector (pSP6ha2FL). The site of 8P6 transcription initiation is marked by the arrow. the SP6 vector-derived sequences incorporated into the 5’ nontranslated region of the transcribed mREA are noted by the zig-zag line. the 5’ and 3’ non-translated regions of the cl-glotiin mRNA are shown as open segments and the (soding region is shown as a filled segment. The positions of the initiation caodon (AJX), termination codon (m) and poly(A) tract (Al@ are noted as are the positions of restriction endonuclease cleavage sites used in generat,ing the fragments (see Materials and Methods). Below this diagram are the fragments generated from the cr-globin cDP;A by a combinat,ion of 5’ terminal Ba/31 digestions and int,ernal restriction rndonuclease digestions (see Materials and Jlrthods). The number t,o the left of each fragment denotes its 5’ terminus. In most cases the 3’ termini are at +dSO (Hind111 site). When this is not the case, the posit,ion of the 3’ t,erminus is noted after the slash. (b) cc-Globin cDNA containing a repeated segment inserted in the 3’ non-translated region. Landmarks are as detailed in (a). A representative cDNA fragment is shown inserted at the Apnl site in the 3’ non-translated region. -4s shown by thtl horizontal arrows. this insert can be

To contirrn that rach antisense insert tiwnid a stern of t,hr predicted size. 5 ~1 of each transcript was added to 100 ~1 of digestion buffer (10 rnM-Tris. H(‘I (pH 7.5) 300 mM-lu’a(‘l. 5 tnM-EDT.4) containing single-strand specific RNases: 2 pg of Rl\;ase A and 0.1 pg of RNase T, (Gibco-RRL. Gaithersburg MT) and Sigma. St T,ouis MO. respectively). The reaction was incubated for 10 min at 25°C’ and terminated by t’he addition of 10 pg of prot,ein” 3 to @5qi, (w/v). then inc.ubatcd an addase h and hDh tional 15 min at Si"(', extracted with phenol/chloroform and precipitated with ethanol. Following drsicca,tion. the reaction products were analyzed on an HO,, wrylamide/ 8 fil-urea gel. (({I 1n vitro to

tmn~latioris

r-(:lobin or a-globin derivative a mi~rococc~al nucleasr-treated

transcripts rabbit

were added rrtirulocyte

inserted in either the antisense or the sense orientation relative t,o the host cDNA. The vector-drrivrd Sal1 site 3’ to thr (~DN~4 was used for linearization of thr plasmid prior to transcription. (c) a-Clobin mRKA containing a - 11 antisense insert. The predicted stem structure formrd by base-pairing between the wdinp rrpion and thr complementjary antisense segment is shown with the connecting loop and the regions 5’ and 3’ t,o the stem represented as single-stranded domains. The position of the 5’ cap is noted by a filled rirclr and thr other landmarks are as described above.

lysatr i/t ,xitro ttxtislatiotl tlrt~ailrd (1,irhhabrr rt

sgstrtti

pwptwl

iw f)wvic~ttsl>

(~1..1984). l3ac.h translation reac*tiott c.ottt,ainrtl 1,-[4,,5-~H IlrwcGw (126 ~‘i/rnmol. Amt~rshattt) and it twistant amouttt of’ /I-globitr tratwript~ as ititrrtral

c.otttt.01 fat, tube-to-tube variatiotl itt translational clfliI.icsttcy. The amount, of tnRIVA add4 \vas detrrmint.tl tj> rwtfioac*tivti itic~orporatiott and was apprc~x. 100 ttg pet I.5 pi t,ranslation. Thr reac4ons warp inc*uhatetl at :I0 ( ’ fi)r I:! min and 3 pl of rac*h rrac:t,ion was thrn analyatvl b> triton-acid-urea (TAIT) acylatnidr grl elrct~rophoresis (Kovrra et ~1.. 197X). The 12 mitt time point was c.hosvtt as translational activity of plobirt trtRrS.4 (both ttatik-e rtltic*ttloc*ytr R1\;A and synthrt,ic tjranscriptjs) was tlrtrrtrrinrtl to be maximal and linear at this timcl and drops off significantly after 15 min (data not shown). I’trdrr thrsv c.otiditions of in c~it,o translation there was tto ac.tivatiott of’ dsf kinase activity (London rt r~l., l!Wi) as rttotritoretl I)y thr translational avtivit!, of t,hr /I-glol)in ttt RSi\ itrtc~rnal c,ontrol prescant in tbac-h rrsac+iott t ubfi (SW atmv,~). This lack of ds/ kinasr ac%ivatiott is fully consistent with t hr short translation ittc.ubation time and thtx high Ievr~ls of dsREA addrd to thr Iysat,r (Rozeu 4 ~1.. 1990). .\ negative c~otttrol rracation lacked rxog:rnous mRXA and a ftositivr control reaf+iort caontainrd 1 pg of normal hutt~ati reti~ulocytt~ RN‘\. All t,ranslat,ions wt~rt~ in thv littr*ut range of RiXA input to protein synthrsis. cc and /I-globin ftrotjvin syntheses wt’re quantified by direct signal tlvtcvtiott from itnalytic~ gels (F’hosF’1-torlmager: Jlol~ulat I)ynatnic:s). The valutl for the translational rficiettc,y of eacah a-glohin drrivativr transcript b+ths adjustrfl for thf, cluatttity of input mRNA (SW abov~~) xnd normalizfvi for thr ac%ual size of thr transcript so that thv value rr~fic~c~tcvl rrlative molar c~on~rtttrat,ioris of the input mRXAs. This valur for the translation was thrn dividrtl bv the amount of’/Cglohin synthesis in the satne reaction tube to at,rivcb at att z :p globin synthesis rat,io. In rac.h rxprrintrttt the 2 : /j synthesis ratio for thr nativr x-glol)itr trattsc.ript \~as tl~+ittrtl al: I.0 and all other values \verv ttortttalizt~tl to this \.1LIUV.

The monosome formation assay was carried out as previously described (Shakin-Eshlrman & JJirhhabrr. 1988). The preparation of a-glohin cL)EAs with graded 5’ drlrtions has been previously drsc*ribed (Shakin KLirbhaher, 1986). The ri’ termini of thr insert,s. as confirmed by direct sequence analysis. are loc*atrd at -21. -14. -i, +i. +18. +26. t43. + 1%. + 177. + 239. +266 and + 288. rlll insert,s rxt,end to a tvmnton ply(A) tail (1118). mRNA/cI)NA hybrids were prepared and analyzed as prrviously described (Shakin-Eshleman & Liebhabrr. 1988). Briefly, oligo(dT)-c,t,llulosr purifird poly(A+ ) human rrticulocytr tnRNA was 13’ end-labrlrd with 15’-321’]p(‘p (cvtidinr 3’5’-1321’lbis~)hos~)hatr) and Tl RNA ligasr. The’ labeled r-glohin rnRXA was thrtt hybridized to several-fold excess of thta indicated r-globin (*I)XA fragment. \vhich had hrrn rrleasrtl front vrvtot srquenc~ and heat denatured prior to hybridizatiott. Addition of furthrr rxvess of cDX.4 insert or vector DIVA had no adverse rffecat on the resultant translational profile (dat,a not shown). Full hybridization of thr r-globitr mRXA to cDNA was demonstrated by its complet,r setiaitivity to Rh’asr H (Ijonis-Keller. 1979: data not shown). Only samples that were lOO(?b hybridized werr used in the binding assay. A total of IO ng of each of the hybridizrd tnRPu’As. as well as a mock (M) hybridized control (c.l)NA excluded from the hybridization reaction), were then incaubated in separate 15 pl rabbit t&culoc~-tr lysate trattslatjion reactions. which werr rlongation arrested b?

t Iit. aclclitiotl of’ attisottty(.itt (Sigtttit. 2 11lRl tillal rYltlc-~ttlt;l tiott). l’hv tvac+ions were prvittcLubattv1 at 1.i ( ’ for I .i mitt tc, eqttilil)rattk thr tc~trrperat ttre. tttRX~\ atlcl~i. ;tttcl f hvt~ tlir). b+f~rf’ irrf~ubated at 1,’ (’ for at1 additic~ttal i.Itttiti (‘ontrol f~sy,rrinirnts tlrrttc3nhtratt~ that I!3 tniti t+ I\ ithitr ass~~mbly atrcl that .tssa~itic thr lirrcvit’ range of tnonosotrtf’ at shortvr or Iongvr time f)fjitlts tlors riot al+f~l thcb txtto of ittr*orJ)orat iott (Shakitt -l%hletttatl X tt1l r3)tttf)aris~~tt 1sith thr (ttott~h~~)ridizetl) fl-globitt IIIRK.Aitt the siftttca n;rtrif)lr~ (intrrnal c~ontrol) It>- verticalI?- sc3tttritrg ~(.li littt~~ ott tlicL Mith a -ofi litwr tlrtisitc~tttt~tf~t~ isf~a paI autoradiographs This ttortttalizat~iott to an itttt~rttal r’otti rc~l wit\ tth~t’). tlonr to c.ontrol for variations in RSA rc~c~c,vc~t.>t’rottt tltci ~radirtit-f)urific,tl rnot~osot~i~~ ftxrtiotis for, (ba(4i s;ttttfslt*.

3. Results

X

full-length

a%plol)in

c.I)N;\.

pSl’Bhcr;!YI,.

extending from the .i’ c*ap addit’ion sitv (position -37) to the 3’ polo tail (.-\]I%) WitS cY~tlstI~lIc~tf4 in the transcription vrvtor. pSP64 (Materials and Methods). This plasmid was linearizetl at a unique ;1paT site in thr 3’ nott-translated rrgiott of’ tjhth cc-globin vI)NA and tletitirfl x-ylobin f4I)N.A f>nents \verv individualI\ inserted at this >itcb in

either the sense or ant~wnw orientatiotr (Fig. 1). Each of thew plasmids was then transc~riiwd to synt,hrsizr a vapped. full-lcwgt h, z-glohitt mRSA or cc-globin derivative rnRS.4. Since all tratiswipts were identical from their 5’ t~rrmini through t)hth initial segment. of the 3’ non-translated region, and WYIY cwrietl since t htl syntheses of all I ranwripts out

in

it

~ornnif~ri

7mC:(5’)ppp(G all transwipts

mix

mastrt

cap nuc~leot idr. were wppetl

[)rimcvi

with

it was assumt~d t ha1 with

t~clual

fd5fGfwf~y.

flirwt I> documented 1)~ a greattar than IOO-fold locwr translat’ional rfif+nf~y of tratIsf*ripts synthesized in thv absence of cap primer attfl a grrntc7 t him IOO-folcl inhibition of translation of t)hc c*aplIefl tritttsc.ril)ts itt the presenw of (‘al) analog (data tlot sl10~1). Full-length /Cglol)in mRSA was t ransf~rilwtl from a full-length fi-glohin c*I)NA (SW’ Materials atid Methods). EfEcient

Each

capping

of these

of the a-globin

trariswipts

t,ransoripts

wxs

synthesized

front

a cI)NA template containing an antisense insert, was predicted to form a stable duplex of defined size and position (example shown in Fig. I(c)). 7’0 cwtfirm

mRNA

Secondary

Structure

(a

E

613

And Translation

1

0 I-(DG$;-“‘T

m

::

RNAse

:

A + T, stems

resistant

nt

(b)

Figure 2. Synthesis and structural mapping of cc-globin and a-globin derivative mRNAs containing repeated segments in the 3’ non-translated region. (a) 321’-labeled SP6 transcript~s. The full-length P-globin, a-globin and a subgroup of the a-globin derivative transcripts were analyzed on an 8 M-urea. C5% acrylamide gel. For display on this gel the amounts of mRNA loaded in each lane were adjusted to (approx.) balance radioactive content. The identity of each mR,NA (see Fig. 1) is indicated abovr the autoradiograph. Earh of the a-globin derivative mRNjAs contains the indicated insert in the antisense orient,ation unless specified as sense (S). DXA size markers (pGEM3 digested with Hi&I) are shown to the left of the gel. (b) RXase mapping of the SP6 transcript,s. To confirm the formation of the predicted RICA duplexes. the transcripts were digested with a combination of single-strand-specific nucleases: RKases A and T,. The resistant fragments were resolved by denaturing acrylamide gel electrophoresis. The lanes on this autoradiograph are labeled as in (a) and 32P-end labeled molecaular mass markers are displayed in the 1st lane (M).

presence of’ t,hese structures, each of the specified transcripts was mapped with a combination of single-strand specific RNases, A and T, (Fig. 2). revealed an RXase-resistant These digestions segment of the predicted size in each transcript containing an antisense-oriented insert and the efficiency of formation of all the duplexes was approxiof signals in (a) mately the same (compare intensity and (b)). Using t*he same digest conditions, the transcripts containing sense-oriented inserts were c~omplrtely RNase sensitive. The duplexes that formed in the transcripts with antisense inserts were assumed to he intramolecular as opposed to intermolecular on the basis of several criteria, (data not shown). (1) The RKase A +T, resistant stems formed immediately upon transcription and reform immediat,ely after heat denaturation. (2) Neither the immediate formation of these RNase resistant stems nor their position-specific inhibitory effect on translation was altered by heat melting and reannealing over a lOO-fold range in concentSration. (3) The major band on non-denaturing acrylamide electrophoretic analysis of the 32Y-labeled mItEA containing t,hr antisense repeat was not altered by heat melting and rrhybridization of a 100.fold the

dilut’ed sample. These data suggest that) the major RNA st,ructure is formed by a zero-order. intramolecular reaction.

in,trttmolecular

duplexes

is position-drpendrllt

The translation efficiency of full-length cc-globin mRNA was compared with that of each a-globin derivative mRSA containing a separate site-specific duplex limited to the coding region. In addition, as a control. a duplex that extended into the 5 nontranslated region ( - 14, see Fig. 1) was also added to this series. In the later case we expected full arrest of translat’ion based on a number of previous studies (Patterson et al., 1977; Hastie B Held, 197X; Kozak. 1980. 1986h, 1989; Privalsky & Bishop, 1982; Liebhaber et al., 1984; Melton. 19%: Spena et al., 19X5: Pelletier & Sonenberg. 1985; Shakin B Liebhaber. 1986; Galili et al., 1986; Parkin it al., 198X: Edery et a,l., 1989). The first set of mRNAs that were st,udied contained antisense inserts that, formed duplexes extending from a common 3’ terminus (+270) to a series of 5 termini spanning positions

-14

to

+ 153 (inserts

-11.

1. 18. 4X. 57.

c~orrlpart~(t \vit tr at1 arl)it,rar>.

LT,

a-GlobIn mRNA u

* 7

d

-

??

$!

2

2

/n v/fro tronslatlon

EL?, -

B - Globln

(0) -_~-- ____ ------------------

-------------.----_

--~------------

__3’ 5’ Extent

of the a-globln

mRNA

duplex

tlW1

ViItlUf’

ivc, x~plohin llt’

I.01

for

tnlhan either of the 18 transcripts. The translations of members of the original cr-globin mRNA series - 14. 18 and 43 were compared with the corresponding duplexes in the deletion series (AK Al8 and A43 mRNAs) as shown in Figure 5. These data reveal that the 25 base deletion in the 5’ nontranslated region has no effect on the translation of the cr-globin mRNAs: the ct43 shows the same increment of t,ranslat’ional activity over 1X as the A43 does over the A18. These data demonstrate that t,he inhibition of translation by these coding region duplexes reflects their proximity to thr :AL\rTcl. (f) The position-dependent suppression of translation by an mRIVA stem rejleets a parallel wrest qf monosome formation, As detailed in the above studies, duplexes that extend to a position + 18 or closer to AUG have the most marked suppressive effects on translation.

,s. .-l l,if4hab~r

tj I ti

A43

(b)

0.0

-14

18

43

Aa

Al8

A43

Transcript (cl

Figure 5. Effect of altering the distance from a coding region duplex t,o the 5’ cap on the translational efficiencies of cw-globin derivative mRKAs. (a) Diagram of the 18 and 43 mRXAs and the A43 mRNA in which 25 vertorderived bases in the 5’ non-translated region have been deleted. The landmarks on the diagrammed mRRiAs are as detailed in Fig. 1 wit,h the zig-zag line representing vector-derived sequences in the 18 and 43 transcripts. The delet,ion of X5 bases from t,he 43 transcript is indicated and the dist,ance from the 5’ terminus of each duplex to the 5’ cap is noted. (b) In vitro translation of the cc-globin. - 14. 1X and 43 mRil;As in parallel with corresponding mRNAs with deletions of 25 bases from the 5’ nontranslated region (Aa, A18 and A43). (c) Relative translational efficiencies of the mRNAs assayed in (b). The means and standard deviations represented in the histogram represents the results from 7 independent experiments.

Since the fully assembled 80 R ribosome covers approximately 14 to 20 nucleotides on either side of ACG (Kozak, 1989: Wolin & Walter. 1988: and included references), this mapping suggests t,hat a critical component of the inhibition relates to the extension of stable duplexes into the 80 S ribosome assembly region. This mechanism would predict

et al.

assemhl>- k)>, wt.11 of that the etfi(.ien(sy of’ monoson-w the transcsripts should parallel its tr;~~lsl>~tic)n;tl a,c+ivity. \l’e have previously desc~ribrd ali (BxIw~~ mental approac*h that pwmit,s a dir.ctc*t asswsrn(‘ll t of’ the effect of I)osit,iori-sl)~,(.i~~,, duplesc~ on n~o~~~~hom~~ fi)rmation (Shakin-Eshlrman k I,iebhaber. 19X8). Irr t.his “hybrid-arrest. of monosomt’ formation” ;rssa> of’ c>l)N.As an mRN.A is hybridized to a serirs covering specified regions. and thr illc~or~)clratic,rl of the mR?iA into the monosome frac%ion of an allisomycin (f%longation) inhibitrd in /*ill translation system is measured. In the present c+astb. :I’ 321j end-labelctl normal human trticulocytp m I( SX (cont’aining both a and j-globin rnRNAs) wah ustld as substrate for monosome assembly. This labt~led rnR;I’A was hybridized to x-globin c~l>?iA fragmcwts covering specific segments of the 5 rlctn-t,rarlslat.c~c1 region and coding regions (Fig. G(a)). Full h>-bridization of the a-globin was documented by R~XWV 13 digestion (see above). The unhybridizrd p-globin mRSA in each sample srrrtld as an internal c,ont rol on R,NA degradation and sample recover>. (see Materials and Methods). These samples ww then incubated in rabbit reticlulocyte Iysate c.c)ntaining t,he translat.ional rlongation lrrhihiior artisornycin and subseyuent#ly fractionated on su(‘rose gradients. The monosome fract,ion of cJac#h reac%tion was coollected and analyzed for intaorporation of the laheled z-globin mRX\;A (for details. see Materials Methods: Shakin & Liehhahcr. 1986; and Shakin-Eshleman & Liebhaber. 1988). A llurn her of t,he oIlSA inserts hybridized to the sc-glohin mRNr\ have 5’ termini ident)ical to those inserted in the :I’ non-translated region of the r-globin transcariptb to create thr intramolwular duplrses (~- 11. 1X. -I-3. 153: Fig. 1(a)). A representative gel analysis of t hr monosome formation reac+ion and a summqv of several independent experiments are SIIOUII it) Figure (i(b) and ((8). respec’tively. The dataa drrrrow &rate that if the mRXA is involved in a duplex that extends quitr c>lose t)o .A[‘(: ( IX or fi1ut.r k)asw), monosomr formation is severeI>- inhibited. If the hybrid exposes more of the rnRNA czodinp srclut’n(~f~ 3’ t,o AUC: (43 or more bases) the monosome formation efficiency increases to levels approximately r-globin rnRNLA. This 60 0,;) of the unhvbridizrd pattern of inhibirion of monosome formation b) (alINhybridization parallels the site-specific t+icfiirric~J of overall translational inhibition du plrxrs from the intramolecular resulting The parallel (compare Fig. 6(c) wit,h Fig. 3(h)). nature of these two assays suggests that. the t#ec~ts of’ the coding region duplexes on mRNil translation are mediatt>d at the lrvel of translation initiatioli.

4. Discussion Alterations in translational efficiencies by general and non-specific perturbations of mRSA secondary structure have suggested a direct> role for secondary structure in translational cont’rol (Tlan 6%Tlan. 1977: Payvar & Ychimke; 1979; Kozak, 1980: Fu rt nl.. 1991). Attempts to define accurately the relation-

mRNA

Secondary Structure

ships between mRNA secondary structures and translation have met with a number of difficulties. A central problem has been an inability to model accurately mRXA secondary structure and to relate specific secondary structures to translational activities. The present study attempts to define the effect of coding region mR?r’A secondary structure on translation by placing extensive and perfectly basepaired intramolecular duplexes at different’ sites within a test mR?;A. Since the primary structures of the 5’ non-translated region and entire coding region remain unaltered. differences in translation can be directly related to the introduction of a highly stable and positionally defined duplex. The data summarized in Figure 3 demonstrat’e marked differences in the ability of specific coding region duplexes to sl:ppress the translation. These differences appear to reflect relative positioning of the duplex in the mRT\;A. (loding region duplexes that extend proximal to. or involve, the m initiation codon (18 and 1i respectively) suppress translation t,o levels t)hat are on average 50/b to 8()6 of normal cc-globin mRXA and are equivalent to the level of suppression hv a duplex that extends into the 5’ non-translated region ( - 14). As duplexes are positioned further 3’ in thr mRNA, translational eficicnq increases and reaches a plateau of 70% with t,he exposure of 85 to 108 bases 3’ of AUG. The contribution of duplex size t’o this effect appears to be minimal. A 70 bp duplex, which extends into the c5’ non-translated region (- 14/56). strongly suppresses translation while a 185 bp duplex beginning at +85 translates at eightfold higher levels (Figs 3 and 4). \Vhen the size of the duplex with a 5’ terminus at +X5 is altered over a 3.5-fold range by truncating or extending it’s 3’ border, translational efficiency is altered by less than 0.5fold (Fig. 4). These data demonstrate that the effect of intramolecular coding region duplexes on translational eficiencg is primarily position-dependent. The ability of coding region duplexes t’o suppress translation relates to their positioning relative to xL’(:. This point is directly addressed by the experiments shown in Figure 5 and the mechanistic basis by which coding region duplexes located proximal to x(: suppress translation was investigat’ed by analvsis of monosome formation (Fig. 6). Previous studies have clrtermined that approximately 14 to 20 bases to either side of AUG: are protected by the 80 S ribosomc during stable monosome formation (Kozak. 1989; \Volin $ Walter, 1988; and references included therein). The +43 duplex would be the first in the series of stem-containing RXAs in which this region is fully exposed. The suppressive effect of the more 5’ duplexes may therefore reflect a steric inhibition of X0 S assembly. The parallel effects of intramolecular and mRNA/cDh’A duplexes on translation and on monosome formation: respectively (compare Figs 3 and 6) supports t’his possibility. Tn addition. the - 14 duplex, which extends into the 5’ non-translated region, may block the scanning of the 40 S ribosomal subunit. Such a possibility is consistent with the inability of the cap-

And Translation

617

binding complex and 40 S ribosomal subunit to destabilize extensive double-stranded segments within the 5’ non-translated region (Pelletier & Sonenberg, 1985; Kozak, 1986b, 1989; Rozen et al.. 1990). Therefore, inhibition of t’ranslation by coding region duplexes in close proximity t.o m may reflect interference with either 40 S scanning and/or 80 S ribosome assembly. The results of the experiments summarized in Figure 3 demonstrate that translational elongation can efficiently destabilize substant,ial intramolecular duplexes that, are situated in the coding region distal t,o the AUC: codon. The primary evidence that translation might be inhibited by such structures comes from the demonstration of discontinuous elongation and/or elongational pausing (Protzek & Morris, 1974: Smith, 1975; T,izardi rt al., 1979; Candelas et (II., 1983) 5’ to areas of mRNA secondary structure (Ran & Jlan. 1977: Pavvar & Schimke. 1979: Vournakis & Yary. 198%: \@olin 8r M’altrr. 1988: Fu et al.: 1991). While such data are intriguing, it has not been possible t’o demonstrate a corresponding functional impact on translation efficiency. If duplexes were capable of blocking the elongating ribosome in a functionally import#ant manner. one would expect a significant decrement in the translation of mR?rTAs containing extensive and perfectly base-paired duplexes over 200 bases in length, such as those synthesized in the current study. Based on the present data. we conclude that if elongation pausing does occur. it does not have a major effect on overall translational etliciency. Although the cc-globin derivative mRN&4s with duplexes at substantial distances 3’ to the AUG: t’ranslate efficiently. their activities are reproducibly 30°6 below that of the control cc-globin mRNA (Fig. 3). I$‘e observe a parallel suppression of monosome formation by duplexes far 3’ to m (Fig. 6). This effect of internal duplexes on translation efficiency is not particular to a-globin mRXX as we have previously demonstrated a similar suppression by hybridized cDSA coding region fragments on b-plobin mRNA monosome formation (ShakinEshleman & Liebhaber, 1988). The mechanism underlying this depression in translation initiation by codmg region duplexes remote from A4 is not defined. A4t least two possible mechanism rnight be considered. The first is that> the highly stable duplex in the mR,PL’X may interfere wit,h potentially important interactions between the 5’ cap and the 3’ poly(A) tail (Monroe & .Jacobson. 1990: Gallie, 1991). Although the coding region duplexes do not extend into the poly(A) tail. they do involve the 3’ non-translated region and may tether the 5’ and 3’ ends of the mRXA in a manner that may interfere with productive translation initiation react’ions. Alternatively, these results may reflect alterations in the higher-order mRNA structure in the region of m critical to efficient monosome assembly. The importance of the native structure in the ribosome assembly region is supported by thr observation that the dimensions and structure of the 80 K assembly site surrounding +axi may in fact vary for

AUG

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(c)

Figure 6. Ribosome-binding activities of 32P end-labeled a-globin mRNAs hybridized to a series of cDNAs with defined 5’ terminal deletions. (a) Diagram of the 32P end-labeled native a-globin mR1L’A (top line) and the set of defined cr-globin cDNA fragments to which it was hybridized. Each of the cc-globin cDXr’$ fragments shown in the diagram extends from a common 3’ terminus (T18) to specified positions within the coding region or 5’ non-translated region (noted to the left of the diagram). The mRNA landmarks and positions are as described above. (b) Monosome formation 32P end-labeled cr-globin mRNA hybridized to defined rDNA fragments (see (a) above). The autoradiograph displays ziel analysis of a-globin mRNA/cDNA hybrids isolated from the 80 S f r-actions of elongation-blocked in nitro translation reactions. The 5’ extent of the cDNA in each mRNA/cDNA hyb r-1 ‘d is indicated above each lane. The positions of the 32P end-labeled c( and P-globin mRNAs are indicated to the left of the autoradiograph. The c( and p-globin mRNAs isolated

m RNA

r\‘econdary

Structure

References

different mRNAs. For example, efficient monosome assembly on the human b-globin mRNA is achieved with

the exposure

of 18 or fewer bases 3’ from m

(Bhakin-Eshleman & Liebhaber, 1988), while exposure of a corresponding region in the a-globin mRK;A is incompatible with efficient initiation (Fig. 6). These results suggest that the imposition of duplexes within the mRNA coding region may perturb an otherwise optimized higher-order conformation of the native mRNA with a subsequent impact on ribosome assembly. The structural determinants of an efficient ribosome assembly site and the nature of the disturbance in this structure resulting from local perturbations in mRSA secondary structure appear to constitute significant variables in translational efficiency. Thr efficient translation of the mRNAs with well within the extensive secondary structure coding region is consistent with previous studies from our laboratory and by others demonstrating that the 80 S ribosome has significant helix-destabilizing activity during translation elongation (Liebhaber et al., 1984; Melton, 1985; Shakin & Liebhaber, 1986; Kozak. 1989). Such an activity would, in fact,. be required for the ribosome to read the primary structure of the mRNA during translation. Although a number of R?;A helicase act’ivties have been described. it is unlikely that’ any of them can account for this activity. The melting activity intrinsic to certain components of the capbinding complex is limited to dest,abilizing relatively short duplexes (fewer than 18 bp in length: Pelletier & Sonenberg, 1985; Kozak, 1!386h, 1989: Rozen it al., 1990) and appears to be discharged from the 40 S subunit at the time of 60 S at,tachment and 80 S assembly (London et al.. 1987). An unrelated RNA editing and “unwindase” activity has been ident,ified in a wide spectrum of cells (Bass Hr Weintraub, 1987; Rebagliati & Melton. 1987: Wagner & h’ishikura. 1988; Wagner of al., 1990) that (#an relax extensive RX4 duplexes via modificat,ion of adenosine to inosine baseh (Bass RWeintraub, 1988; Wagner et al., 1989). However. this activity appea,rs to be limited to t,he nuclear compart)ments (Wagner et al.. 1990) and is not present in reticulocyte lysate (LVagner cf al., 1990: and our unpublished data). In the absence of other relevant act,ivities. it. is reasonable to conclude that the melting of caoding region duplexes is associated with. and may represent 1 an intrinsic property of the elongat,ing 80 S rihosomr (Liebhabrr it nl., 1984: Shakin 8 Liebhaber, 1986).

619

And Translation

Bass. H. L. & A’rintraub, H. (1987). A developmentally regulated activity that unwinds RNA duplrxrs. Cull, 48. 607-613. Bass. 1s. I,. & Weintraub, H. (19%). An unwinding ac+ivity that, covalently modifies its double-stranded R?iB substrate. C’ell, 55. 1089-109~. C’andelas. (i.. (‘andrlas. T.. Ortiz. A. Pr Rodriguez, 0. (1983). Translational pauses during a spider fibroin synthesis. Biochrm. Riophys. Rw ~‘oruncun. 116. 1033%1038. Donis-Keller, H. ( 1979). Sit? specific enzgmatic~c~leava~e

of R?iA. Srrcl. Acids Kes. 7. 179P192. Ed~ry. T.. Prtryshyn. R. & Sonenbrrg. .1ctiration of double-stranded RNA

N. (1989). dependent

kina.sr (ds1) by the TAR, region of’ HIV-I mRSA: a novel translational control mechanism. (‘~11. 56. 303 312. Fu. l,.. Ye, R.. Browder. L. IV’. & ,Johnston. It. X. (1991). Translational potent’iation of messenger lfor mRSA translation fractionated together with rabbit rrticuloc‘vtr initiation factor 3 c~oniplrx. I’ror. Snt. dctld.

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lT.S.A.

74,

23252329.

Tserrntant. I). & Firrs. W. (1980). Secondary structure of mRX;A and efficiency of translation initiation. Oerw, 9. l-12. KoLak. 11. (1980). Influence of mR?;h src*ondary strurturr on binding and migration of 40 S ribosomal sutlunits. (‘PI/. 19. 79-90. Kozak. 11. (19841. Point mutations (alost, to the A-\I’G initiator codon affect the efficient>- of translat,ion of in P~PO. Snture fJ,onrlon). 308. rat preproinsulin “41 246. Kozak. 11. (1986~). Point mutations define a sryuenre flanking the ‘it!C: initiator codon that modulates translation by eukaryot,ica rihosomrs. ('~11. 44. -‘8:I (mock). (c) Quantitative comparison of monosome formation activit)ies. The graph represents a quantitative summary of monosomr formation assays as in (b). The monosome formation activities were calculated as described in Materials and Methods and are compared w&h mockhybridized a-globin mRNA (100%). Each data point represents an independent analysis of a particular hybrid. The data points corresponding to each mRNA/cDNA hybrid are plotted at the position along a-globin mRNA corresponding to the Ti’ extent of the cDNA used (horizontal axis). A vertical line indicates t,he + 1 position on the mRS=\. The positions of the cap ((‘AI’). initiation codon (Am). termination codon &j&j) and poly(.-\) tail ((A)n) are indicated.

-

I’rotw. .I. & Jlorrts. .A. .I. (I!fi4). (:rl (,tirotttilto~raI)lti(. 240 analysis of trastwit globin cahnins. .I. Niol. (‘hew Kozak. >I. (l!M). I,ea,drr length and srcwndary Nol. (‘rll tttodulatw m R,ri.A function.

struc*turcl Kid.

8.

“737 -‘il4. Kozak. 11. (1989). (!ircumstattws and trieehatiisms ot inhibitiott of translation by secondary structuw in ciuc,aryotica trtRX.As. Mol. (‘~11 B’iul. 9. 5134~-5112. Lirbhabrr. S. .-I. 8: (‘ash, F. E. (1985). IAoc~us assignment of’ cc-globin structural mut,ations hv hybrid-wlectrtl translation. ./. (‘lit/. Inwst. 75. tiG70. Lirbhabrr. S. A.. (‘ash, F. E:. & Shakitt. S. H. (1984). Translationall> associated hf,lix-tiestabiliiit~~ acstivity in rabbit) rrtiwlocyte Iysatp. ,/. Iliol. (‘hrrx 259. I .‘,.5!G I X02. Lizartli, I’. >I.. Mahtlavi. V.. Shields. I). & (‘andrlas. (:. (1979). 1)iscontinuous translation of silk fibroin in a rrticuhyvte cell-frrr system in ittt,ac.t silk gland culls. I’ror. Sul. .-Id. Sri.. 7‘.~S.cl_ 76. 621 I 621:?. London. I. hl.. Levin. I). H.. Matt,s. R. l,.. Thomas. N. S. H.. I’etryhyn. 1~. 8r Chett. ,l. .J. (1987). R,egulation ot prott~in synthesis. In Thr Enzymes (Hoyer. P. 1). B Krebs. IX. C;.. ~(1s). vol. IX. pp. 3:59-M). Acaademic l’rrss. Nrw York. Maniatis. T. Fritsch. E. F. & Sambrook, J. (198%). :~llolrrulrtr

f ‘Ion iny.

.-I

twltoratory

Manucrl.

Spring Harbor Laboratory I’ress. Harbor. SY. Mrlton. I). i\. (1985). Injected anti-srnse wily block mrssenger RNA translation Sat.

Jloldavc~.

Awd.

Sri..

K. (19%).

IY.S.A.

82.

ICucaryotic,

t ‘old

(‘old

Spring

KSi\s

spwiti-

in uiw.

J’roc.

4.594-4600.

Ray.

H. K.. T,awson. ‘1’. (:.. Kramer. .J. (‘.. (‘latiaras. .\I. H.. (irifo. .I. A.. Abramson. Irtilizrd frog ~‘ggs rr%v& MI RSA duplrs un\vinding activity. f ‘~11. 48. 599 Wi. (‘. ~9 I~OIIIJ~. T. 12’ ( IOiX). Rovrra. (i.. Magaria.n. Rwoltttiott of hemoglobin subunits 1~).c,It,c,t,rol)llor~,sis tn ac.1~1 utw polyacrylamidr gels cxontaining Tritott S-100. Anal. Biochrn/. 85. Wfi SIX. Rown. F.. Edr~r~~. 1.. L)ev~r. T E.. J1rrric.k. \I- ( ‘. R Sonrnbrrg. N. (1990). llitlitwtional R?U’A helicasr activity of’ ruc~aryotic~ translation initiatiott fwtors 4A and 4l? Mol. f’rll nior. 10. I I31 Ill-c. Sangrr. I:.. Sic,klett. S. Sr (‘oulsr~~. I’\.. (I!)ii]. I)NA seclwncittg lvith c,haitt-trrminating inhibitors I’roc,. Srrl. .-Irrlrl. Sri.. I~.S.,J. 74. .‘,463-d467. Shakin. 1;. H & IAirbhabrr. S. -\. (l!Mi). I)t~stal)ilizatio~t of trrrssrt~grr R~.-\:c~otrtItlr~nit~~~tat~~ 1)X1\ tluplt~xw by, the cslongating 80 S riltosotne .I /iid (‘hw~ 261, 160 I X Itio2T,. Shakin-Eshl~,ttt;tn. S. H. clr I,irbhabc~r. S. .1 rI!)XX). Ttlflrrrtlm~ of’ tluplexw :$’ to thca tnItN.\ ittitMioti codor~ on thr rfEciet1c.y of tnonosomt’ l’ormatiott. Hiorh~nt

istry.

27.

397S

398,.

lb-148.

protein

synthesis.

.-lnrru.

I%/*. Kiochrnc. 54. 1109-l 149. Monroe, I). 8 Jacwbson. A. (1990). mRSX polg(A) tail. a 3’ rnhanwr of translational initiation. Mol. (‘ill Hiol. 10. 344l- 34%. Mot+. F.. Starrk. H. & Godrt. .I. (1986). r-thalassetnia due to the deletion of nucleotidcs - 2 and - 3 prwrdinp thr r\IyC initiation codon affects tranwlation e!?icirtwv bot,h in t?tn, and in Gw. .VcA. Acids

tirs. 14. 3%79-‘3292. Muthukrishnan. S.. Both. (:. CV.. Furuichi. 1-. & Shatkin. :\. .I. (1975). r,’ Terminal ‘i-methylguanosine in rukaryotic mRXA is reyuirrd for translation. Srrturr

(London),

255. 35 40.

I’arkin. N. T.. (‘c,hrn. IC:. A. Darveau. .I.. R,OSHJI. C’.. Hasrltittr. L!‘. 8: Sonenberg. IV. (1988). Mut.ational analysis of the 5’ non-wding region of human immunodefic~iency virus typtx I: effwts of srcondar? structure on translation. EURO .J. 7. 2831-2837. I’attrrson. K. 11.. Roberts. R. E. Br Kuff, E. L. (1977). Structural g~nr identification and mapping b) 1)X;.\ tnR,N.L\ hybrid-arrrstrd crll-free t,ranslation. f’ror.

Sot.

.-lmd.

JVci..

1’.9.,1.

76. 4370

4374.

I’ayvar. F. & Stahimkr. R. T. (1979). Mrthylmrrcur~ hydroxide enhancement of translation and tranwrip tion of ovalbumin and c.onalbumin mHS.A’s. .I. Rid. (‘hrm.

254.

Sownberg. S. (19X8). (‘al) binding protrins of rarrkar.votic. meswngf~r RX:\: func&tions iIt initiation ant1 c,orttr’ol of 35. t,ranslatiott. I’qr. S//r/. .3 cirlh Rrs. Nol. Hid. 17-c “07. si pena. .\.. I)rauw. K. & I~obberst.ritt. I