Bacterial expression of rat liver succinyl-CoA synthetase α-subunit

J. Mol. Biol. (1991) 219, 165-174

Bacterial Expression of Rat Liver Succinyl-CoA Synthetase a-Subunit Factors that Contribute to Blocked Translation of Transcripts Encoding a Mitochondrial Signal Sequence David G. Ryan and William A. Bridger Department of Biochemistry University of Alberta Edmonton, Alberta T6G 2H7, Canada (Received 13 September 1990; accepted 28 January

1991)

This study comprises a detailed evaluation of factors that’ are necessary to achieve high levels of expression of eukaryotic proteins in bacterial systems. We attempted to express a rat liver cDNA clone encoding the precursor to the a-subunit of succinyl-CoA synthetase in an Escherichia coli expression system, without success. Removal of the region encoding the mitochondrial signal peptide (115 nucleotides) allowed efficient expression of the mature protein. This nucleotide sequence was shown to block expression at the level of translation. Two regions within this fragment were able to block t’he expression of other genes such as E. coli la&. Inhibition of expression was due to the close proximity of these inhibitory sequences with the translation initiation region (TIR). Insertion of a spacer between the inhibitory sequence and the TIR relieved the block in translation. Analysis of the 115nucleotide fragment identified sequences capable of extensive base-pairing with the Shine-Dalgarno and surrounding sequences. Such secondary structures are capable of blocking the formation of competent translation initiation complexes. Keywords:

translation;

expression;

ribosome binding 1acZ fusions

1. Introduction Succinyl-CoA synthetase (SCST) catalyzes a step of the tricarboxylic acid cycle in the mitochondrial matrix. In mammals, the enzyme is a heterologous dimer of a and /?-subunit.s that are encoded in the nucleus and synthesized in the cytoplasm as larger containing precursors mitochondrial signal sequences (Wolodko et al., 1986; Henning et al., 1988). These precursors are processed to the mature subunits by removal of the signal sequence following translocation through the mitochondrial membranes (Majumdar & Bridger, 1990). The mature subunits then are assembled within the matrix to produce the active dimeric enzyme. t Abbreviations used: SCS, succinyl-CoA synthetase: SC&a, succinyl-CoA synthetase a subunit; SCS-pa, precursor to succinyl-CoA synthetase a subunit; PAGE, polyacrylamide gel electrophoresis; RBS, ribosomal binding sequence: Ig, immunoglobulin; TIR, translation initiation region. Restriction sites designated with an asterisk (e.g. ,Vcol*) are those constructed by site-directed mutagenesis.

sequence;

mRNA

secondary

structure:

Succinyl-CoA synthetase provides an excellent model for investigating the process of mitochondrial import and assembly of a protein with a heterologous subunit structure. The process of denaturation and refolding of this enzyme in vitro has been studied extensively (Pearson & Bridger, 1975; et al.: 1988; Wolodko & Bridger, 1987; Nishimura Khan & Nishimura, 1988), demonstrating that full activity may be recovered from reconstituted mixtures of the previously denatured a and B-subunits. To that end, we wished to determine the effect of the signal sequence on the refolding process, and to demonstrate possible association of newly synthesized precursor with chaperoning proteins (Deshaies et al., 1988; Chirico et al., 1988). A cDNA clone (SCS-pa) has been isolated from a rat liver Agtll library that encodes the mature asubunit together with an N-terminal 27-residue signal sequence (Henning et aZ., 1988). To investigat’e its folding properties, we have attempted to express this clone in Eseherichia coli using wellcharacterized, high-level expression systems. No detectable level of protein was obtained using clones

I 66

Il.

(i.

Ryan

and

(b) PT7-6

(C) hSCS 19-l 490 I E

045 164 A 11 1 VNHK SIgnal s.equcncc

697 I B Open reading frame

891 ,017 1066 12c4 ,II , H P E t

Figure 1. (a) plJCl9W, showing the translation initiation sequence cloned between the Hind111 and BumHI s&s. Insertion of genes between the HamHI and EcoRI sites renders them capable of translation in E. coli. S/D. Shine--Dalgarno region. (b) Cassettes were transferred as HindTTI EcoRI fragments into corresponding sites in pTT-6, thus placing them under the control of the T7 $10 promoter. (c) The restriction map of 1SC19-1. a rat liver c*I)1\‘A clone. The reading frame, which includes the signal seyuence, runs from the NcoT site at position 0 to position 1017. Restriction sites are as follows: Nu’?(NcoI; 0); E. (EcoRI, 490 & 1204); H, (HinPI, 45 and 891); K, (KpnT. 164; 13. (BarnHI, 697); P, (PstI, 1066).

nptimally placed downstream from a strong translation initiation sequence. These results were unexpected in view of the fact that the corresponding E. coli cl-sequence (with about 70% sequence ident.it,y) could be expressed to SOY/, of total cell protein (Buck & Guest, 1989). In this paper we have evaluated factors that are necessary to achieve high levels of expression in a bacterial system. In particular we describe the use of a phage T7-coupled in viva expression system that allows specific labeling of the expressed protein to analyze our results. We have examined the S(Spa coding sequence with regard to inhibition of We conclude that the presence of the expression. encoding the signal sequence blocks region expression at the level of translation. Furthermore, we propose that such sequences interact directly thus initiation region with the translation preventing active initiation complexes from forming.

2. Materials and Methods (a) Bacterial

strains

and vectors

A1I genetic manipulations were performed as described by Maniatis et at. (1982). E. coli strains JM109 and GM48 (Yanisch-Perron et al., 1985) were used in the molecular cloning work. Expression was carried out in a DHl host

IV. A. Hridgrr

(Hanahan. IYW). Thtl plasmid I)(.(‘19 (Vieira &, .\ll~ssing. 1982) was modified by insertion of a wnsensus t r.;tnslation initiation sequence (see Fig. I (a)) to give ply(‘I!)\\‘,. Clvn(~ fusions were made in this modified plasmitl nntl MW~ verified by sequence determination. Construcsts Marc’ tht>rr transferred as Hind111 -EcoRT fragments to thr expression vector pT7-6 (Fig. 1(h)). The complete open reading frame of S(X-pa was clonrtl into pUc’lSW, as an NcoI-EcoRI fragment, whereby the SCOT site was blunt-end ligat,ed to the HnmHl site in thr vector. Tn addition, partial mung bean nucleaatb trratm~~nt of the X:coI and HallcHI sites followed by ligat.ion resulted in a + I framtl shift calonecontaining an additional nu(.lr+ tide after the ATG start, cxodon. A 2nd Scot* sitr NW created at position 115. This shorter SCOT* /?,‘wI blunt end ligation into both the XdeI and ~S’all restriction sites on pTi-7. This results in placing the S(Wpc( c,oding sequence 0 and 13 amino acid rrsidues. respec*tiv(Bly. from thp AT(: start &on in the vector. (b) Expres.sion The plasmids pGPI-2. PT?-6 and ~‘1‘7-7. useti in thr coupled TS expression system. were gifts from I>r Stan Tabor of’ Harvard University. The plasmid pGPl-2 contains the T7 RNA polymerasr gene under a heatinducible promoter. pT7-6 is used to (alone the desired gene under the control of a T7 410 promoter. Procedures for exclusive labeling of plasmid-encoded proteins using the T7 RKA polymerase/promot,er system were rssentially those described by Tabor & Richardson (1!385).

Site-directed mutagenesis was performed by the method of Zoller & Smith (1987) with modifications by Kunkel et al. (1987). All mutations were ma&: in M13mp18. Mutants were identified by restriction enzymr analysis and confirmed by dideoxy sequencing @anger rt al., 1977) before transfer bac>k into the expression vector pT7-6. (d) &VA

isolation

and hybridizution

To measure levels of transcript produced in expression systems, cells were grown at 28°C with aeration to A600 = 0.5. A portion (1 ml) of this culture was induced by shifting to 42°C for 15 min. Rifampicin was added to a final concentration of 0.4 mg/ml and the culture was incubated for 30 to 45 min more to deplete host RKA. The RNA was then isolated by the hot phenol/SDS procedure of von (Gabair ct rrl. (1983) and subjected to

Expression of Succinyl-CoA Northern blot analysis on GeneScreenTM membrane according to t,he manufacturers recommendations (NEK Research Products, DuPont). Northern blots were probed with an XcoI-EcoRI fragment of SCS-pa that had been labeled with [35S]dATPaS by the random primer method (Feinberg & Vogelstein, 1983). Alternatively, the cells were pulsed with i3H]uridine (10 pLCi/ml)for 10 min prior to RNA isolation. The relative rates of RNA synthesis were then determined by purifying RKA from indured cells and measuring incorporation of tritium.

SI>S/E)olyac,rylat,litie gel electrophoresis (PA(;E) was run using the m&hod described by Laemmli (1970). fi-(ialactosidase activity was measured according to the procedure of Miller (1972).

3. Results and Discussion This work was prompted by previous unsuccessful attempts to achieve expression of the SCS-per clone as an unfused open reading frame downstream from a strong promoter and ribosome binding sequence. 1)etection of expression involved subjecting total cell lysates to SDS/PAGE analysis followed by either protein staining. immunoblotting. or a.ut.oradiography upon induction of the system. The T7 polymerase/promoter system introduced by Tabor & Richardson (1985) represents a substanmore

sensitive

sysf$em, and

lends

itself

Tao

detailed analysis of factors that can affect efficient expression of foreign genes in E. coli. Tn particular. since host RNA polvmerase can be inactivated by addit,ion of rifampic’in, t,ranscripts produced under the control of the T7 promoter can be selectivelv allowing enriched, excslusive labeling wit,h j 35S]methionine of expressed proteins. Thus, even very poorly exprtassed genes can be detected in total cell lpsates by autoradiography of Sl)S/P.L\GE gels.

We rr-examined the expression of the complete SCX-pa clonr using t:he T7 system. The SCS-pet open reading frame was placed optimally downstream from a strong consensus ribosomal binding sequence (RBS) in the plasmid pT7-6. Cells containing this clone together with the plasmid, pGPl-2, were pulsed with [ 35S]mrthionine following induction. No protein was detracted from autoradiography of a total

cell lysate

subjected

to SDS/PAGE

(Fig. 2,

lanes 3). As a control. the SW-cr from E. coli and its companion fi-subunit were well expressed under t’hese conditions (lane 1). This is consistent with the report of Buck B Guest (1989), which shows that. the bacterial subunit (which lacks a signal sequence) can be effectively overexpressed. In view of the fact that E. coli SW-r is highly homologous to mature rat liver SCS-cl (70 oo sequence identity; see Henning et al., 1988). we focused on the region encoding the N-terminal signal sequence as a possible explanation for the block in expression of SCS-per. Removal of t,he first, 165 basr-pairs (encoding the entire signal

167

Precursor

sequence and a short region of t,he mat,ure protein) in efficient expression (lane 4). The protein was estimated to be of the expected size. and was immunoprecipitable with anti-SCS-cr IgG (Lane 8). Other workers have reported similar difficulty in expressing precursor proteins that can be overcome bv deletion of some or all of the signal sequence (i’tsumi et al., 1988; Vernet et nl., 1989: Schrank r?f al.. 1989; Kaderbhai et al., 1990). results

it)) Expression

(e) Al iscrllaneous

t,iallv

S@btase

block is attributahlp

nucleotide

to

srquenw

It has been suggested that the presence of the signal peptide may inhibit the folding of the mature protein (Randall & Hardy, 1989). If this were the vase one could rationalize the absence of expression as a result of rapid degradation of an unfolded, protease-sensitive precursor molecule. 1~1 the present case, however, we believe that the absence of detectable lower molecular weight degradation products argues against’ proteolysis as a potential explanaLion. We therefore wished to determine whether the observed block in expression was due to the nucleotidr sequence or to the amino acid sequence encoded in the 165 base-pair region. A simple frame shift would distinguish these, while preserving tfhr nucleotide sequence between the frame shift borders. We therefore constructed a clon
of expression of translation

is at thr

Since Ihe expression block had been established to be attributable to the nucleotide sequence, we reasoned that it. could be operative either at. the level oft ranscription or translation. Most expression systems in cotnmon use (including the T7 polymerase/promot.er system) are based on the ability of a strong promoter t,o drive transcript,ion at a high level without. regard to the nature of t,he gene transcribed. Nevertheless, we measured the transcription efficiency from the T7 promoter by the ability of expressing cells to incorporate [ 3H]uridine into RNA under rifampicin treatment. Rates of transcription were compared in the non-expressing and expressing cslones (with the 164 base-pair deletion).

2

IWO

3

4

5

6

ECORI

KPn

II If

A

1 1 k

1

164 1

Exmession

Sign01 sequence

Lane 1 -

u =29,600

E. co/i UP

Lane 2 - pT7-6

Iv,

p=41,400

M,

++++

-

Control NC0

Lane

3-

pT7-6

I+-

-

Km Lane 4 - pT7-6

+++

/+ NCo Frame shift

Lane

5

-

pT7-6

k3& cI+----”

-

Stem-loop ~~~ removed

Lane

6-

Lane 8 -

pT7-6

+/--

-

As lane 4, but subjected to immunoprecipitation (anti-a IgG) ++t

Figure 2. SDS/PAGE of total cell lysates subjected to autoradiography. Clones w-err examined for their ability t,c) produce protein using the exclusive labeling technique. The Figure is a schematic outline of the clones used. T,anr 1 shows E. coli succinyl-CoA synthet,ase c[ and b-subunits (29,600 (29 K) and 41,400 (41 K) M,. respectively). T.tint~ % represents a control pT7-6 without insert,. Lanes 3 to 6 and 8 are different constructs srque~lce (for further details, see the text). RBS, ribosome binding sequence.

Both of these incorporated similar levels of label into RNA following induction (see Fig. 3). The levels were comparable to those of the positive control (pT7-6). This incorporation was resist.ant. to to showing it bl? rifampicin treatment, T7-controlled, whereas the negative control, pUPIlacking a T7 plasmid, showed very low incorporation. Jt must be borne in mind that mRNA decay can play an important role in bacterial gene

containing

rat liver

SCS-p

coding

expression (von Gabain et al.. 1983: Helasco rt (11.. 1986). Expression of eukaryotic genes in E. coli can also be regulated by polynucleotide phosphorylase and RNase I; mutants deficient in these enzymes exhibit increased stability of foreign mRNA (Hautala et al., 1979). Northern blots identified transcript produced in both expressing and nonexpressing systems when probed with SW-a DNA (not shown). These results lead us to conclude that

Expression

of Succinyl-CoA

KP

__.

Synthetase

A scs

169

Precursor

19--l

, 164 Expression

pGPI-2/pT7-6 pGPI-2/pT7-6 pGPl-2/pT7-6

Ws-7 Km

Figure 3. (‘omparison of the rates of incorporation of F3H]uridine into new-l!- synthesized RNA in cells containing expression plasmids. Above the Table is a scheme showing the 2 XX-pa const,ru&s tested for RKA synthesis. Rates werp measured for cells with and without rifamnirin treatment. The negative control was pCI’l-2 only. while the posit,ivr for IO min f&wed by isolation of R<4 c*ont,rol contained pT7-6 in addition. Cells Gere pulsed with ‘H]uri&nr

the transcripts are not significantly degraded and that the inhibition of expression is due to a block in translation. (d) m RNA

secondary

structure

Failure of a t,ranscript of a foreign gene to be translated in E. toll: is not surprising, given the complexity of the translation process and the multitude of mechanisms that bacteria have devised to regulate this event (for a recent review. see McCarthy 8r Gualerzi, 1990). The role of mRNA secondary structure in the control of translation is well documented. Elements of secondary structure have been shown t,o affect the accessibility of the Shine-Dalgarno sequence and the AUG start codon of transcripts (Hail et al., 1982; Tessier et al., 1986). Expression of foreign genes is often adversely affected by sequences at the 5’ end of the transcript,? and this is believed to be due to the ability of these sequences to base-pair with the nearby translation initiation region (TIR) (Schoner et al., 1984; Tessier et aE., 1984. McCarthy et aE., 1988). With this in mind, we examined the SC&pa clone, looking especially at t,he inhibitory region near t.he 5’ end. Among several elements of secondary structure predicted for the sequence, we noted a very stable potential stem--loop at t,he extreme 5’ end of the coding sequence. with a free energy of -22.7 kcal (1 cal = 4.174 ,J). The AUG start codon was sequestered into this st,ructure (Fig. 4(a)). The existence of a stable stem-loop was confirmed by our observation that primer extensions of incompl&ely heatdenatured RNA samples appeared to stall at a point close t,o the 3’ end of the structure. We thought it possible that the involvement of the AUG start codon in such a structure could lead to a complete block of translation. To test this, six residues centrally located along the 5’ arm of the stem were replaced with non-complementary nucleotides, while the encoded amino acid sequence was main-

tained, thus effectively disrupting the secondary structure (Fig. 4(b)). Th e mutant’ sequence was then analyzed for its ability to be translated. There was no change in the inhibition (Fig. 2; lane 6). It would appear that the block in translation is not a.ttribut,able to the presence of this secondary stru&ure. It must be borne in mind, however. that the altered sequence may itself exert a negative influence on translation.

GCC

GCC u u C

G C c

i c C-G c= c G=C

G C C=Gc GsC GZC

i (&

/

c

G-C I c A=U C=G G=C

A=U C=G Erg C'G

C= G

C :

C 1:

Mutation C

C

U=A G’C

U=A G=C

Start

U

UT

cc

u I!

OG:-22.7

kcol

&-4.8

codm

i0)

C

start codon

cc kcal f b!

Figure 4. Prediction of mRNA secondary structure using the program of Zuker & Stiegler (1981). (a) Representat,ion of the strong stem-loop region found at the very start of the SCS coding sequence. The AUG start c*odon found at the base of the stem is boxed as indicated. (b) Illustration of the disruption in the secondary structure following mutation of 6 residues (shaded box) with a resultant decrease in free energy.

170

D. C. Ryun and W. A. Hridger

ffin P

I

k

Signal

sequence

Expressiort Lane 1 - E co/i $3

+t+

(control) NC0

Lane 2 -

pT7-6

I/---

-

Lane 3- pT7-6

IF--

-

It--

-

//----

-

Lane 4 - pT7-6 NC0

(Deletion)

Lane 5 - pT7-6 ~~~ Lane

removed

6 _ pT7-6 p@J ‘----+I-E co/i a

Lane 7 - pT7-6 NC0

Lane 8 -

pT7-fj

Figure 5. SDS/PAGE gel of total cell lysate following the inserts indicated in the scheme above. (See Materials

(e) Sequences encoding the signwl peptide block translation I,ow resolution mapping of the 165 base-pair sequence was performed in an effort to specify more precisely the minimum sequence requirements for inhibition of translation. At the 5’ end of the gene.

+++

c/a*

lz!J’E-+-

_

autoradiography. and Methods

The cells were transformed

for further

details.)

Abbreviations

by pT7-6 having as for Fig. 2.

45 nucleotides can be removed with no relief of the expression block (Fig. 5, lane 3). Hut when an addt,ional 70 nucleotides were deleted the remaining transcript regained the ability to be expressed (Fig. 5, lane 4). Since this result suggested that the crucial sequence mediating translational inhibition resided between residues 45 and 1 Ifi. we produced a

Expression ?f Succinyl-CoA

Synthetase Precursor

171

C/a* scs-pa

P-Galactosldase activity

1

puc19 w,

put 19:

590

put 19(1-164):

320

put 19(1-115):

115

put 19(45-164):

312

ATG

1

put 19(45-115):

JM103 host:

334 Undetectable

Figure 6. Schematic representation of the la& fusions with parts of the first 164 base-pairs of SCS sequence. Both lue TTR and the consensus TIR are in frame with the downstream la& gene. The lac TIR was removed by cloning the fusion from the consensus TIR to the end of the la& into pT7-6. B-Galactosidase activity produced by these fusions was measured by the method of Miller (1972). Activity is given in units/ml culture.

construct with an internal deletion of 65 residues (45 to 110). Somewhat surprisingly, transcript from this construct was still blocked in translation, indicating that residues 1 bo 45 are capable of blocking expression independently. This result prompted reinvestigation of the mRNA secondary structure mutant with the 65 base-pair block deleted. The results (Fig. 5, lane 6) show that this construct also failed to produce translatable transcript. The 115 base-pair stretch is able to downregulate the expression of other genes that are normally expressed well. The SCS-c( gene of E. coli (normally expressed at high levels) was fused in frame to the 3’ end of this sequence, and the resulting fusion exhibited complete block in expression (Fig. 5, lanes 7 and 8). The overall result shows that both sequences, 1 to 45 and 45 to 115, are able independently to autoregulate their own gene expression at the translational level. (f) Translation

proximity

initiation is inhibited by the of sequences to the TIR

In an effort t.o develop a simpler system for detecting inhibition of expression, we constructed a series of LacZ fusions. These were constructed in pUC19 using the coding sequence for the fi-galactosidase cl-peptide and activity was monitored in JM109, a strain carrying a deletion of the u-peptide. Fusions were created with all or parts of the 165 base-pair region (Fig. 6). All of t,hese gene fusions were capable of producing /I-galactosidase activity. The activity produced from these fusions in three cases was approximately half of that from the positive control (pUCl9) suggesting that the /l-galactosidase activity of the resulting fusion pro-

tein is not seriously perturbed by the presence of the additional peptide sequence. The fourth fusion (1 to 115) gave fourfold lower activity, reflecting the fact that the SCS-pa portion of the fusion protein was in a different reading frame. These results were unexpected. In some way, the fragments of the 165 base-pair region no longer inhibit translation. We noticed that the EacZ TIR and the first five amino acid residues of the la& gene upstream from the fusion site were perfectly in-frame with the N terminus of the SCS-pallacZ coding sequence. Such constructs would therefore allow translation initiation from the EacZ TIR to produce functional fusion protein. The foregoing results do not distinguish between initiation at the Zac TIR and at the TIR proximal to the inhibitory sequence, but all previous experiments would lead us to assume that t,ranslation initiation remains blocked at the proximal TIR. We removed the gene fusions from the influence of this upstream ZacTIR and placed them, together with their TIRs, downstream from the T7 promoter pT7-6. All clones, with the exception of produced no the positive control pT7-la&, fl-galactosidase activity and thus had regained the ability to block translation. We conclude that effective translation initiation occurs only at the Zac TIR pUCl9 fusions, and that the consensus TIR proximal to SCS-po: is still completely blocked in both constructs. These results would suggest that it is the proximity of the TIR to the inhibitory sequences that inhibits translation initiation at this site. This result is reminiscent of that of Kaderbhai et al. (1990), who found that the addition of a second TIR followed by four amino acid codons upstream from the first overcame a block in translation.

41 K-++

29 lx--+

EX_DreSSiOn

Lane 1

E Cdl ap

++++ Nqo

Lane 2

pT7-7 13 amino

Lane 3

pT7-7

J fs@EJ-7 NC0

Lane 4

I+

-

I&-

++

lb--

+++

acid residues

pT7-6

NCO*

Figure 7. Analysis of expression of complete SC?+peeconstructs is placed at varying distances from thr 7’1 K. Lanr 2 SCS sequences placed immediately downstream from the TIR region of pT7-7. Lane 3 shows the effect of deletion of IS amino acid residues from the SCS-pee sequence encoded down&earn. The positive control in lane 4 is ~)T5-6 c*ontaining the truncated SCS from NcoT* to E&RI. represents

(g) Insertion of a leader sequence following AlJC: overcomes the block in translation,

the

If proximity to SCS-per renders the consensus TIR ineffective, then increasing the separation could he expected to relieve the translation block. To test this. we have used pT7-7, a vector that contains the T7 410 promoter (as does pT7-6) plus the T7 gene 10 ribosome binding site and its AUG start codon. followed by a multiple cloning site. We placed t.he complete SCS-per coding sequence into pT7-7, 13 amino acids downstream from the initiation codon to ‘1’7 gene 10 (see Fig. 7). Thirteen amino acid residues (39 nucleotides) was thought to be sufic:irnt spacing on the basis of the la& fusion c+onstruets. As a control, the SCS-pa sequence was c~loned immediately adjacent to the TTR. The two constructs were tested for expression using the T7 labeling technique. The construct with the 13 amino acid residue leader sequence was expressed (Fig. 7. lane 3) giving rise to a protein with an additional molecular weight of 1500. The control construct, as showed blocked expression (Fig. 7. expected, lane 2). This experiment confirms that it is the proximity of the TTR to the SCS-pa sequences that is responsible for the complete inhibition of translation

initiation.

4. Concluding Remarks We have shown that expression of a rat liver cL)NA clone encoding the precursor t.o the cc-subunit of SCS was blocked at the level of translation. Sequences encoding the extreme 5’ end were entirely responsible for the effect. Two stretches within the first 115 nucleotides encoding the signal were capable of blocking translation sequence independent.ly. A spacer of 39 nucleotides inserted between tjhe TTR and these inhibitory sequences relieves the block in expression Since there is a need for the sequences to be closely linked to the TIR, we speculate that these sequences may be involved in direct intramolecular base-pairing interactions with the TTR. l’pon inspection, we find that the 115 nucleotide region has a very high G+C-content (62’::) madr necessary by the need to encode specific residues in the signal peptide. Four candidat,e sequences werp tjo the identified that show complementarity Shine-Dalgarno sequence. We have carried out a series of secondary structure predictions and in every case where inhibition was observed. the predicted mRNA fold shows the Shine-Dalgarno sequence to be involved in a region of extensive base-pairing. The sequestering of this ribosome

Expression of Succinyl-CoA binding sequence in stably folded mRNA may prevent the formation of competent translation initiation complexes. These results are in accord with other reports regarding variations in translation efficiency (Tessier et al., 1984; Looman et al., 1986; Schneider & Beck, 1988). In fact de Smit & Van Duin (1990) pointed out that very small differences in base-pairing stability can result in dramatic variations in translation efficiency. There is a point of wide applicability to be made here: namely, that for effective expression in E. coli the sequence at the extreme 5’ end of foreign genes must be optimized so as not to interfere with the structure of the translation initiation region. One effective way of dealing with this problem is to make use of vectors that express foreign genes as fusion proteins, joined in-frame to the C terminus of a highly expressed bacterial gene. It has been widely held that the use of vectors that produce fusion proteins is advantageous because it offers a level of stability and protection from degradation to the foreign protein. One must also recognize an additional advantage to this strategy: possible interference with translation initiation is obvint,ed. We thank Drs 12’. T. Wolodko and .J. H. Weiner fot insightful suggestions. This work was supported by a grant (MT-2805) from t,he Medical Research (‘ounoil of Canada.

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Feinberg, A. P. $ Vogelstein. B. (1983). A technique for radiolabrlling Dr\‘A restrict,ion endonuclease fragments to high specific ac%ivitg. Anal. Hiochem. 132, 6- 13. Hall, M. X.. Cabay. ,I.. IXbarbouillB, M. $ Schwartz. $1. (1!)82). A rolr for mRNA secondary structure in the c*ontrol of translation initiation. Nature (London), 295. 616--61X. Hanahan. D. (1983). Studies on transformation of Eschwichia roli with plasmids. J. Mol. Biol. 166. 557~ 575.

173

Synthetnse Precursor

Hautala. *J. A.. Bassett. c’. I,.. Giles, N;. H. & Kushnrr. S. K. (1979). Increased expression of a pukaryotic gene in Escherichia coli through stabilization of its messenger RriA. Proc. Sat. Arnd. Sci., I-.S.A. 76. 5774-5778. Henning. W. I).. ITpton, C.. Majumdar. K., McFaddrn. (:. & Bridger. II’. A. (1988). (‘loning and sequrnring of the cytoplasmic precursor to the x subunit of rat livrr suc~inyl-CoA synthetase. f’roc. Sfrt. Ad. Sri.. I’.:; a..* 4 85 1472%1476. 1 Kaderbhai, M. A.. Hr. M.. Beeehey. R. B. K: Kaderbhai. ?I‘. (1990). (‘o-expression of a precursor and the mature protein of wheat ribulosr- I .5-t)isI)hosphat,r c.arboxvlasr small subunit from a single grnr in Each.er;chirr coli. 11X.4 (‘rll Hiol. 9. 1 l-25. Khan. I. A. 81 Sishimura. ,J. S. (1988). Xativr-like int.rrmediatr on the folding pathway of Escherichin roli sucGivl-CoLA svnthetasr. .J. Riol. (‘IwII. 263. L’l52-il58. ’ Kunkel. T. .\.. Roberts. *J. I). & Zakour. R. :\. (1987). RalJld end &ic+rnt site-directed mutagenesis wit,hout l)hrnot,ypic* selec+on. ,bfPthOd.S ,hZ?/~KJ~. 154. 365 3X”. Larmmli. I-. K. (1970). (‘Iravage of structural proteins during the assembly of the head of bacteriophage T-l. .Vature

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Edited by J. Karn