THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Val. 260, No. 28, Issue of December 5, pp. 15163-15167 1985 Printed in Zj.S.A.

Inefficient Translation of T7 Late mRNA by Bacillus subtilis Ribosomes IMPLICATIONS FOR SPECIES-SPECIFIC TRANSLATION*

(Received for publication, March 21, 1985)

Paul W. Hager and Jesse C. Rabinowitz From the Department of Biochemistry, University of California, Berkeley, California 94720

Bacillus subtilis 30 S subunits inefficiently recognize initiation sites in mRNAs from Gram-hegative bacte- ria, but they are able to efficiently recognize initiation sites in mRNA derived from Gram-positive bacteria. McLaughlin et al. (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291) have suggested that B. subtilis ribo- somes require a strong Shine-Dalgarno sequence for translation initiation. To test whether this criterion is sufficient to explain the translational specificity of B. subtilis ribosomes, T7 late mRNA, which contains strong Shine-Dalgarno sequences before many of the late genes (Dunn, J. J., and Studier, F. W. (1983) J. Mol. Biol. 166,477-535), was translated in vitro with both Escherichia coli and B. subtilis ribosomes. The identification of several of the in vitro products upon gel electrophoresis indicated that B. subtilis ribosomes recognize correct translation initiation sites in late T7 mRNA, but they do not translate these products effi- ciently. Competition experiments demonstrated that late T7 mRNA does not inhibit B. subtilis ribosomal translation of B. subtilis derived mRNA (from the bac- teriophage 429). It is concluded that strong Shine- Dalgarno sequences may be necessary in B. subtilis translation initiation sites; however, additional deter- minants of initiation which differ from those found in the translation initiation sites of E. coli mRNAs must exist.

The initiation of protein synthesis by procaryotic ribosomes involves the selection of an appropriate site on the mRNA by the 30 S subunit of the ribosome (1-3). In general, ribosomes derived from Gram-positive bacteria translate homologous mRNAs (i.e. Gram-positive derived mRNAs) efficiently, but translate mRNAs derived from Gram-negative bacteria inef- ficiently (4-12). This species-specific translation implies that Gram-positive derived ribosomes and mRNAs are function- ally distinct from those of Escherichia coli. Since E. coli ribosomes translate mRNA derived from Gram-positive bac- teria, it has been assumed that such mRNA contains, at a minimum, determinants of translation initiation which are similar to those found in mRNA from E. coZi (13). One of the features of mRNA important for its recognition by E. coli ribosomes is found in the “ribosome binding site” which

* This research was supported by Grant AM2109 from the National Institute of Arthritis, Metabolism, and Digestive Diseases of the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

includes an initiation codon, a Shine-Dalgarno sequence, and an appropriate spacing (“window”) between these two (1-3, 14,15). The Shine-Dalgarno sequence consists of a polypurine stretch of variable length which is located 5’ to the initiation codon and is capable of base pairing to the 3’ end of the 16 S rRNA (16).

McLaughlin et al. (13) have suggested that the Shine- Dalgarno complementarity required by Bacillus subtilis and other Gram-positive ribosomes is significantly greater than that required by E. coli and other Gram-negative ribosomes, and is required for species-specific translation. The sequence information for over 40 Gram-positive derived translation initiation sites that has appeared since its formulation sup- ports this hypothesis since all of the sites contain “strong” Shine-Dalgarno sequences (17). The “strength” of the Shine- Dalgarno sequence was estimated by determining the free energy of formation of the most stable double helical complex between the 3’ end of the 16 S rRNA and the Shine-Dalgarno sequence according to the rules of Tinoco et al. (18). The Gram-positive derived translation initiation sites have Shine- Dalgarno sequences with free energies of binding with an average value of -16.7 kcal/mol (standard deviation of 2.3), as compared to E. coli translation initiation sites which have an average of -10.9 kcal/mol (standard deviation of 3.4).

The known E. coli translation initiation sites (19) include a number which have strong Shine-Dalgarno sequences with calculated free energies of binding that overlap those of B. subtilis (17). The E. coli phage T7 contains an unusually large number of these strong ribosome binding sites (20). To deter- mine whether a ribosome binding site composed of a strong Shine-Dalgarno sequence with an appropriately placed initi- ation codon is a sufficient determinant for translation by B. subtilis ribosomes, we tested mRNA prepared from T7 DNA for activity with an in vitro B. subtilis translation system. We decided to test the late region of T7 for this purpose because of its complete characterization and abundance of strong ribosome binding sites and the ease of identification of protein products using mutants and because there are few polar effects in T7 gene expression (20). We find that B. subtilis ribosomes do translate authentic T7 late proteins, although at a mark- edly reduced level compared to E. coli ribosomes. There is some correlation between the strength of the Shine-Dalgarno sequence and the relative expression of the protein by B. subtilis ribosomes; however, there must be other features of a Gram-positive translation initiation site which are important for efficient translation which remain to be elucidated.

15163

15164 Translation of T7 Late mRNA by B. subtilis Ribosomes

EXPERIMENTAL PROCEDURES AND RESULTS~

The late region of T7 (i.e the region transcribed by the T7 RNA polymerase) contains several genes that are preceded by strong Shine-Dalgarno sequences with4ree energies of binding close to the average value of -16.7 kcal/mol characteristic of Shine-Dalgarno sequences derived from Gram-positive oga- nisms. Fig. 1 shows the response to E. coli and B. subtilis ribosomes to the addition of T7 late mRNA and to $29 mRNA. While B. subtilis ribosomes translate the 429 mRNA, they are practically inactive on the T7 late mRNA, despite the relatively strong binding energies of these sequences. The relatively poor translation of $29 mRNA by E. coli ribosomes would seem to be in contrast to the earlier reports of efficient translation of this mRNA by E. coli ribosomes (11). There are two factors which are responsible for this apparent change in the translatability of $29 mRNA. First, we have used [35S] methionine rather than [3H]lysine to increase the sensitivity of our fluorograms, and B. subtilis, but not E. coli, ribosomes predominantly synthesize the gene 6 product (also referred to as the 13.9-kDa protein) (11,21). The sequence of $29 gene 6 (21) shows that it has an unusually high methionine/lysine ratio compared to the other open reading frames of the major early region of $29 (22). Since E. coli ribosomes do not translate this protein, they appear less active when using [35S] methionine. Second, earlier work used completely homologous transcription and translation systems, while in experiments reported here, B. subtilis components have been utilized as much as possible which has previously been shown to affect the absolute levels of translation by E. coli ribosomes (11).

Although B. subtilis ribosomes do not translate T7 late mRNA efficiently, products of this translation were identified following polyacrylamide gel electrophoresis (Figs. 2 and 3).

I I I I I I 1

A. E: coli ribosomes

Transcription Reaction (,d)

FIG. 1. Protein translation as a function of added mRNA. A, incorporation of methionine by E. coli ribosomes in response to 629 mRNA and late T7 mRNA. Reactions of 30 pl contained either $29 transcription reactions (0) or late T7 transcription reactions (01, 17 pg of E. coli ribosomal salt wash, 80 pg of B. subtilis S-150, and 0.7 A,,,, units of E. coli ribosomes. B, incorporation of methionine by B. subtilis ribosomes in response to $29 mRNA and late T7 mRNA. Reactions of 30 pl contained either 629 transcription reactions (0) or late T7 transcription reaction (U), 11 pg of B. subtilis ribosomal salt wash, 200 pg of B. subtilis S-150, and 0.6 AZG0 unit of €3. subtilis ribosomes.

Portions of this paper (including “Experimental Procedures,” portions of “Results,” and Figs. 2-4) are presented in miniprint at the end of this paper. The abbreviations used are : DTT, dithio- threitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-863, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

The relative amounts of each protein made by E. coli and B. subtilis ribosomes were quantitated by scanning fluoro- grams with a densitometer (Table I). B. subtilis ribosomes show greater relative translation of proteins that have strong Shine-Dalgarno interactions (note 3.5, 11, and 14) and less relative translation of those which have weaker Shine-Dal- garno interactions, as compared to E. coli ribosomes. The exception to this is seen with protein 9, which does not have a strong Shine-Dalgarno interaction but is translated by B. subtilis ribosomes at an increased relative amount as com- pared to E. coli ribosomes. These results indicate that while B. subtilis ribosomes do not efficiently translate T7 mRNA they may prefer initiation sites that have strong Shine-Dal- garno sequences.

DISCUSSION

T7 late mRNA is inefficiently translated by B. subtilis ribosomes despite the strong Shine-Dalgarno sequences in many of these mRNAs. Thus, although strong Shine-Dal- garno sequences appear to be necessary for translation of mRNA by B. subtilis ribosomes (23), such sequences are not sufficient to allow the efficient translation of E. coli phage mRNA by a system containing B. subtilis ribosomes. The products of the low levels of translation include the same proteins made by E. coli ribosomes, so B. subtilis ribosomes appear to recognize the correct initiation sites on T7 late mRNA; however, B. subtilis ribosomes prefer different initi- ation sites than E. coli ribosomes. The translation of T7 late mRNA by B. subtilis ribosomes could be inhibited at any stage, but the ability of B. subtilis ribosomes to translate $29 mRNA following preexposure to T7 late mRNA (Fig. 4) suggests that B. subtilis ribosomes inefficiently bind to initi- ation sites on T7 mRNA. Poor translation from what is otherwise a good initiation site is analogous to some recent examples of translational regulation (23-26). It is possible that there is a factor which inhibits the translation of heter- ologous mRNA by B. subtilis ribosomes. Unlike gene 32 pro- tein (24), or the ribosomal proteins (25) which inhibit trans- lation of specific mRNAs, such a factor would have to be quite nonspecific. In addition it would have to block translation for B. subtilis ribosomes but not E. coli ribosomes, since the components of the translation systems are interchangeable (11,12).

The inducible resistance genes derived from Gram-positive organisms appear to use a different mechanism for transla- tional regulation (26). The mRNAs are capable of at least two mutually exclusive conformations. In the repressed state the Shine-Dalgarno is sequestered in a stem structure and would

TABLE I Relative expression of some of the T7 late proteins by B. subtilis ana‘

E. coli ribosomes Expression of identified proteins is given as the ratio of each

protein relative to the total ,incorporation of the reaction, based on densitometry of fluorograms of sodium dodecyl sulfate gels. Also indicated are the binding energies (AG) in kcal/mol for the interaction of the translation initiation site with the 3’ end of the 16 S rRNAs of B. subtilis and E. coli.

Protein E. coli AG to B. subtilis AG to product ribosomes E. coli ribosomes E. subtilis

2.5 0.09 -12.8 0.05 -14.0 3.5 0.08 -18.8 0.18 -21.2 5.5 0.30 -11.8 0.12 9

-11.6 0.02 -14.6 0.16 -12.8

10 0.10 -12.8 0.04 -14.2 11 <0.02 -17.8 0.06 -21.2 14 <0.02 -17.8 0.05 -21.2

Translution of T7 Late mRNA by B. subtilis Ribosomes 15165

be less accessible to the ribosome. A second conformation, stabilized when a ribosome stalls while translating the leader peptide, exposes the Shine-Dalgarno and the initiation codon facilitating translation of the mRNA. T7 translation initiation sites might be blocked in secondary structures, hence poorly translated by B. subtilis ribosomes. However, our computer analysis of T7 translation initiation sites shows them to be generally free of stable interactions which would mask the Shine-Dalgarno and initiation codon. In the functional sense, these sites are accessible since E. coli ribosomes do translate these mRNAs. It is possible that E. coli but not B. subtilis ribosomes are able to read through particular secondary struc- tures. Such a function could be performed by the E. coli ribosomal protein S1, which has no homologue in B. subtilis (27-29) and which stimulates translation of E. coli derived mRNA by Bacillus stearothermophilus ribosomes (30). How- ever, E. coli S1 does not stimulate translation of E. coli derived mRNA by B. subtilis ribosomes (11).

Our preferred interpretation of our observations is that E. coli and B. subtilis translation initiation sites share some common elements but differ at least with respect to specific features c-f these elements. An analysis of 42 translation initiation sites derived from Gram-positive organisms sup- ports this view (17). The common elements include the Shine- Dalgarno sequence and its spacing to an initiation codon. The Gram-positive derived Shine-Dalgarno sequences are “stronger” than the average E. coli sequence, but the spacing to the initiation codon is similar. The center of the Shine- Dalgarno sequence is usually found about 10 bases upstream of the initiation codon. Although an initiation codon is an invariable part of a translation initiation site, B. subtilis and other Gram-positive organisms use non-AUG initiation co- dons more frequently than E. coli (29% to 9%) (17, 19). E. coli ribosomes appear to utilize additional sequence prefer- ences within the ribosome binding site (31, 32); for example, these sites tend to be A-U rich. The Gram-positive derived initiation sites accentuate this characteristic. A collection of B. subtilis and other Gram-positive derived mRNAs contain 42% A residues for a 50-base region around the initiation site (33).

In summary, B. subtilis translation initiation sites differ from E. coli sites, both with respect to the strength of the Shine-Dalgarno and possibly to sequence preferences within the translation initiation site. A strong Shine-Dalgarno se- quence is not a sufficient signal for efficient translation of a mRNA by B. subtilis ribosomes, but is probably a necessary one. Additional features of a strong B. subtilis translation initiation site remain to be elucidated.

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Continued on next page.

15166 Translation of T7 Late mRNA by B. subtilis Ribosomes Supplementary Material to

Inefficient Translation Of T7 Late mRNA by 8. subtilis Ribosomes; Implications Of Species Specific Translation

by

Paul W. Hager and Jesse C. Rabinowitz

EXPERIMENTAL PROCEDURES

"_ Bacterial Strains and --- T7 phage wild type and the amber mutants 2-64, 3-29, 4-20, 5-28, 6-147, 7-213, 8-11, 9-17, 10-13, 11-37, 12-3, 13-149, 14- 140, 15-31, 16-9, 17-290, 18-182, 19-10, all originally described by Studier

University of California, Berkeley. T7 strains LG-3 (a deletion spanning the (341, were obtained from M. J. Chamberlin, Department of Biochemistry.

genes 1.1-1.3) (34). 2.5427a (35). and the amber mutants 3.5-lys13a (36), 5.5-B64a (35). were obtained from F. W. Studier, Brookhaven National Labora- tory, Upton, New York. The following E. & strains were used, E. & B/lr

preparation of T7 RNA polymerase. were obtained from M. J. Chamberlin. E. used for the growth of T7 wt and LG3. and E. & DG-156. used for the

w. Studier. coli 011.. used for the growth of the other T7 mutants, was obtained from F.

Materials--Chemicals were purchased from the following sources: Cesium chloride, biochemical grade, Gallard-Schlesinger; spermidine trihydro- chloride, ATP, CTP, phosphoenolpyruvate, lysozyme (chicken egg white) Pyru- vate kinase (rabbit muscle), rifampicin, Tris, sodium deoxycholate, and DTT Sigma Chemical Corp.; E. coli Stripped tRNA, Boehringer Mannheim Biochemi- cals; polyethyleninine ( 5 0 % ) . Eastman; bovine serum albumin, Armour Pharma- ceutical co.: ammonium sulphate, special enzyme grade, Schwarz/Mann Tnc.;

Chemists, lnc.; polyethylene glycol 6000, Fisher Scientific; all gel electro- trichloroacetic acid, Mallinckrodt; EDTA (disodium salt), MCB Manufacturing

phoresis reagents, Bio-Rad; GTP (tetrasodium salt) and UTP (trisodium salt) P-L Biochemicals; [4-32P1cTP (3000 Ci/mmol), 135Slmethionine (1000 Ci/mmol) I

and aqueous counting scintillant, Amersham; phosphocellulose e-11, whatmani Heparin-agarose (37).

" T7 RNA Polymerase-- T7 RNA polymerase was purified by the method Of Kassevetis and Chamberlin (381.

T7 --- T7 phage was grown, and plaque assays were performed, essentially

phage or with a tangential flow concentrator (Millipore Corp.). Concentrated were concentrated either using polyethylene glycol 15%) to precipitate the

pha e were banded twice in CsCl gradients. Titers were generally 1.5 x 101a/A?60unit on the permissive host 5. & 011'. The amber mutants showed reversion on the nonpermissive host of less than 0.01% that On the permissive host except for 10-13. 10-13 had a reversion to wild type of 1%. which is not uncommon since gene 10 is the major coat protein of the T7 phage particle (F. W. Studier, personal communication).

" T7 DNA-- DNA was isolated from phage particles as follows. CsCl purified phage were dialyzed versus 10 mM Tris-C1. pH 8.0. 5 mM MgClz, 0.5 M NaCl overnight, and then diluted to an A260 of approximately 30. Phage SOlUtions were extracted with phenol (Saturated versus 1 M Tris-C1, pH 8 ) . then extracted with phenol/CHC13/isoamyl alcohol 24:24:1. and then extracted with CHCl /isoamyl alcohol 24:l. The solutions were then dialyzed sequentially agaiist 11 10 mM Tris-C1, 1 mM EDTA, 0.5 M NaC1, pH 8. 2) 10 mM Tris-Cl. 1 mM EDTA, pH 8. 3) 10 mM Tris-C1, 0.1 mM EDTA, pH 8. The DNAs had final Con- centrations between 0 . 5 ana 1 mg/ml.

In Vitro RNA Synthesis-- T7 late mRNA was prepared as described by Kassavetis and Chamberlin (38) except that ribonucleotides were increased to 1 mM and T7 RNA polymerase l1.200 nU/mg) was added to 0.16 mg/ml and one Unit Of activity

material. Reactions of 50 or 100 ul contained 0.1 mg/nl DNA, 40 mM Tris-C1 represented the incorporation of 1 umole/min of CMP into acid-precipitable

1pH 81. 8 mM MgCl , 5 mM DTT. 4 mM spermidine, and 1 mM ATP. CTP, GTP, and UTP. Reactions wgre stopped With the addition Of EDTA to 8 mM and were stored frozen at -70 OC. Under these conditions, a 100 "1 reaction contain-

precipitable material. For the preparation Of late mRNA from the DNA of T7 ing DNA from wild type T7 phage incorporated 16 "moles of CMP into acid-

mutants. reactions were reduced to 50 "1 and the incorporation of CMP into acid-precipitable material ranged between 4 and 8 "moles. e . RNA Polym- erase (190 mu/mg) was kindly supplied by Martin Schmidt and M. J. Chamberlin. For the preparation of T7 early mRNA the reaction conditions were identical to the T7 RNA polperase conditions except that E. & RNA polymerase WBS

added to 65 u g h 1 and reactions were stopped by the addition of EDTA to 8 mM and rifampicin to 20 ug/ml. Under these conditions a 100 ul reaction incor- porated 12 nmoles of CMP into acid-precipitable material. 8. s a RNA polymecase (29 mU/mg) and 629 DNA 137) were kindly supplied by Mark Roberts of this laboratory. RNA was synthesized as previously described I371 except that ribonucleotide concentrations were increased. Reactions of 100 u1 con- tained 100 nM Tris-C1, 10 mM MgCl , 1 mM DTT, 1.6 mM spermidine, 160 mM KC1, 1 mM ATP, CTP, GTP, UTP, 0.1 ng/m: 629 DNA and 0.3 mglml 8. subtilis RNA polymerase. Reactions were stopped by the addition of EDTA to 10 mM and rifampicin to 20 ug/nl. Under these conditions a 100 ul reaction incor- porated 12 "moles of CMP into acid-precipitable material.

a i described by Studier (35). POT the purification of phage, phage lysates

In vitro RNA-directed Protein s "thesis-- Protein synthesis reactions were zr;iedouts- t o a p r e v r o u s description (5) in a 30 ul reaction volume containing 67 mM Tris-C1 pH 8.0. 0.1 M KC1, 50 mM NH4C1, 0.83 mg/ml 5.

tRNA, 6.3 mM phosphoenolpyruvate, 17 ug/ml pyruvate kinase, 0.1 mM EDTA, 12 mM p-mercaptoethanol, 0.5 mM GTP, 2 mM ATP, 12 mn Mg(0Ac) , 10 uM 135Slmethionine with a Specific activity of 20-50 cpm/fmole,2renaining 19

protein of 8. subtilis ribosomes (22 ug) or E . & ribosomes (17 us) 139). amino acids each at 50 uM, initiation factors were supplied from Salt wash

0 . 6 AZs0units of E. & or B. Subtilis Vacant couple ribosomes (12 Pmoles) (39). 0.1 mg B. subtilis s-150 or E . S-150T IS) , 7.5 ul of T7 or 629 2 vitro transcription reaction was added directly to these reactions to supply =and the reactions were incubated at 37 Oc for 20 min. Reaction were stopped with the addition of 10 ul of 0.25 M TriS-C1, pH 6.8, 50% glycerol, 5% SDS . FDC the measurement of radioactive incorporation into protein a 10 u1 aliquot was removed and precipitated with the addition of 1 ml of 5% tri- chloroacetic acid. The sample was placed on ice for 5 mi" and then was heated to 90 OC for 10 mi" to deacylate charged tRNA. The sample Was cooled and the precipitate collected on a glass fiber filter (24mm). The filters were washed with 6 ml of cold 5% tCiChloroaCetiC acid, then 5 m1 of 95% ethanol, then dryed under a heat lamp and counted in aqoeous counting scin- tillant. For analysis by SDS-PAGE, p-mercaptoethanol was added to 1% and the sample was heated to 90 OC prior to loading on the gel.

el Electrophoresis-- Polyacrylamide gel electrophoresis using SDS and a diSContinuoUS buffer system (40) with an acrylamide/bis-acrylamide ratio of 37:l was performed in a Slab electrophoresis cell. Gradient gels containing 10-209 acrylamide with a stacking gel of 5% acrylamide were used. For fluorography the gels were fixed in 10% acetic acid 25% isopropanol and then

were adjusted to give reasonable exposures overnight at -90°C using Kodak XAR soaked in 1 M sodium salicylate (41) before drying. Sample volumes loaded

an A540=0.16 to give a linear response to radioactivity 1421. Densitometric film. Film which was to be used for densitometric tracing was preflashed to

with a 3390A reporting integrator from Hewlett-Packard. traclngs were made with an EC910 densitometer from E C Apparatus equipped

Determination of Protein-- Protein determinations were made using either the dye-binding procedure of Bradford (43) or the Lowry method (44) with bovine

cipitation by 10% trichloroacetic acid as the first step. serum albumin as a standard. The Lowry method was modified to include pre-

RESULTS

TO identify Some of the major protein products of T7 late mRNA the DNA of T7 mutants were transcribed with T7 RNA polymerase and then translated with E.

translation products made by E. ribosomes. the major late pro- ducts of T7 are those of genes 2.5, 3.5. 5.5, 8, 9, 10, and 17 1201. Iden- tifiable from the fluorograms are the products of genes 1.3, 2.5, 5.5, 9, 10, 11, 14, and 17 (1.3 protein is missing in the LG3 deletion, the 2.5 protein from the 2.5-JZ7a Strain has an altered mobility on SDS gels. and other pro- teins are missing due to their amber mutations). Gene 10 produces 2 protein products identified as 1OA and 1OB. D u m and Studier have concluded that 108 arises from a frameshifting which occurs occasionally during the translation of 10A (201. Protein 6, from strain 6-147, should be shortened from it5 nor- mal molecular weight of 39,995 by 24 amino acids (20). There is an altered band at a molecular weight of about 40,000 which would indicate that this band represents protein 6. However, this band corresponds, both in intensity and location, to protein 10A. Close examination also Shows that the mobility of 106 is slightly altered in lane E of Pig. 2. This isolate Of 6-147 has probably picked up a second nutation which has altered the mobility of both IOA and 10B. therefore protein 6 is not identiEied on these fluorogrnms.

" coli and 8. subtilis ribosomes. Fig. 2 shows fluorograms of SDS gels of the

-17- , lOB\

-2.5'

'14- -11-

FIGURE 2

Electrophoretic analysis of proteins translated by E. & ribosomes using T7 late mRNA.

Translation of T7 Late mRNA by B. subtilis Ribosomes 15167 T7 late mRNA was prepared from wild type and mutant Tl DNA as described

under Experimental Procedures and added to translation reactions as indi- cated. La= 5 contained transcription reaction minus DNA. Translation

mental Procedures. Labeled proteins were separated on lo-20% polyacrylamide assays containing E . ribosomes were performed as described under Experi-

gradient gels and visualized by fluorography.

are identified in Fig. 3. The proteins which are most easily identified Some of the products of the translation of Tl by 8. subtilis ribosomes

include 1.3, 2.5, 9, 10. 11, and 14. In the cases of 3.5 and 5.5, the pro- tein band at the expected location is missing, but there appear to be other changes in the pattern of translation products as well. There are two prom- inent protein bands from the B. subtilis in vitro translations which have not been identified (found between 3.5 and 5.5 in Fig. 31. When equal amounts of acid-precipitable counts are compared, B. subtilis and E . ribosomes prefer to translate different products (see Fig. 3, 5 and i].

Since the B. subtilis ribosomes do translate several of the T7 proteins, they It is not clear why B. subtilis ribosomes translate Tl mRNA poorly.

recognize the Correct initiation sites for these proteins. This inability to efficiently translate Could be due to poor initiation of translation 1l.e. weak binding to the initiation regionl. Alternatively, initiation complex formation could proceed normally followed by a block at a later point in the

bind to Tl mRNA in Unproductive complexes, these complexes might prevent the translation as Suggested by Sharrock & (45). If 8. subtilis ribosomes

ribosomes from translating a mRNA presented at a later time. Such a competi-

late mRNA and then adding 629 mRNA as shown in Fig. 4 . T7 late mRNA lor the tion experiment was performed by peeincubating E. &tb ribosomes with Tl

transcription reaction) does not inhibit translation of 629 since E. subtilis

poration. E.. S- ribosomes are not inactivated by preexposure to Tl ribosomes with 629 sRNA or with 629 plus T7 late mRNA give the same incor-

late mRNA, since addition of 629 mRNA at 5 rnin results in immediate incor- poration at a rate similar to the initial rate using 629 mRNA alone. The absolute level of incorporation for this competition assay is lower than for 629 mRNA alone, but this is probably a consequence of the loss of activity these ribosomes display after 15 mi" 133). Thus, the relatively inefficient

binding to Tl late mRNA ln nonproductive complexes. translation Of T7 late mRNA cannot be explained as B. subtilis ribosomes

e- 10- 11- 14-

I I

A

- 3.5. - 5.5 -

Electrophoretic analysis of proteins translated by B. subtilis ribosomes using T7 late mRNA.

under Experimental Procedures and added to translation assays as indicated.

assays containing B. subtilis ribosomes la-h, and &-"I or E. coli ribosomes (1 and i l were performed as described under Experimental Procedures. Labeled proteins were separated on 10-20% polyacrylamide gradient gels and visualized by fluorography.

T7 late mRNA was prepared from wild type and mutant Tl DNA as described

" Lanes a and i contained transcription reactions minus DNA. Translation

-y 4

@' T7icp29 added at 5min

T7 Late 0' I I I I

0 5 10 15 20

Time (mid

FIGURE I

Translation of Tl Late mRNA and 629 mRNA.

Amino acid incorporation by 8. subtilis ribosomes with Tl late mRNA and 629 mRNA in a competition assay. The translation reaction was initiated

mRNA. FOE reactions which contained 629 mRNA, 3ul of 629 transcription rex- using ribosomes, ribosomal salt wash, and 5-150 from 8. subtilis and T7 late

tion was added directly.