translation initiation at non-aug triplets in mammalian cells* · expression in cultured monkey...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 9, Issue of March 25, pp. 5031-5035,1969 Printed in U.S.A.

Translation Initiation at Non-AUG Triplets in Mammalian Cells* (Received for publication, May 9, 1988, and in revised form, November 22,1988)

David S. Peabody From the Department of Cell Biology and the Cancer Center, University of New Mexico School of Medicine, Albuquerque: New Mexico 87131

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In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D. S. (1987) J. Biol. Chem. 262, 11847- 11851). In order to assess the capacity of the mammalian translation apparatus to initiate at other non- AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide. The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter. Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase. Incorporation of [96S]tRNAp into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine. The mutant dhfr sequences were also inserted into the mammalian expression vector SVGTS for expression in cultured monkey cells. The hierarchy of relative translation efficiencies was similar in vivo and in vitro.

AUG is the usual translation initiation codon in all species studied so far. However, it has long been recognized that other triplets are sometimes utilized in bacteria. For example, a comparison of the sequences of more than 250 Escherichia coli genes (1) revealed that AUG is nearly always the initiator triplet, but natural examples of initiation at several non-AUG triplets do exist (also reviewed in Ref. 2). GUG, UUG, and AUU have all been shown to serve as initiation codons in some cases. There are also several examples of mutants with functional AUA initiation codons. It is well documented that AUG, GUG, UUG, and UUU are able to stabilize the binding of initiator tRNA”“‘ to ribosomes i n uitro (3-5). By contrast, the eucaryotic translation initiation apparatus has been thought to exclusively utilize AUG triplets. This was the conclusion of an extensive genetic analysis of revertants of mutations in the initiation codon of the yeast cycl gene (6) and of the initial failure to find non-AUG initiation codons among the many known sequences of eucaryotic mRNAs. However, more recent evidence is changing this view. There are at least three natural examples of this phenomenon. The first was described by Becerra et al. (7) who demonstrated

*This work was supported by Grant DMB-8510683 from the National Science Foundation. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

that one of the capsid proteins of adeno-associated virus is initiated at an ACG triplet. Curran and Kolakofsky (8) and Gupta and Patwardhan (9) have reported that synthesis of C’ protein of Sendai virus is also initiated at an ACG initiation codon. In addition, ACG has been shown to direct translation initiation with a mutant form of the bacteriophage T7 0.3 gene i n vitro (10) and with a mutant version of the mouse dhfr gene i n vivo and i n vitro (11). In most of these cases it has been demonstrated that methionine is the initiating amino acid. Another example was recently reported by Hann et al. (12) who have found that a second product of the human c-myc gene results from translation initiation at a CUG triplet upstream of the initiator AUG. Several other triplets are competent for translation initiation of mutant mRNAs in yeast (13, 14), but usually at very low levels, and there is currently no information as to whether methionine is utilized.

In our previous study (11) ACG was shown to be an efficient initiation codon i n uitro when it resides within a favorable sequence context as defined by the rules of Kozak (15-17). The ability of ACG to serve as an initiation codon caused us to wonder whether other non-AUG triplets might also be capable of functioning in translation initiation. As a prelimi- nary approach to this problem, we have constructed each of the possible single nucleotide variants of the dhfr’ initiator AUG. The resulting nine mutants (including the previously studied ACG) have been compared for their ability to direct the synthesis of DHFR. The results indicate that several other non-AUG triplets are competent for translation initiation.

MATERIALS AND METHODS

Construction of Plasmid Recombinants and Mutagenesis of the dhfr Translation Initiation Site-The construction and properties of the ACG mutant have been described previously (11). Similar methods were employed in the present study in which the initiator AUG of mouse dhfr was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. Oligodeoxyribonucleotides were synthesized on an Applied Biosystems model 380A DNA synthesizer and used to mu- tagenize a fragment of dhfr cloned in M13mp8. The structures of the mutants were determined by DNA sequence analysis (18), and the complete dhfr sequence was reconstructed in pSP64 (19) for in vitro transcription/translation studies. The mutant dhfr sequences were also inserted into the SV40 vector, SVGT5 for expression in cultured mammalian cells (20). The sequences and nomenclature of the mutant initiation sites are shown in Fig. 1.

I n Vitro TranscriptionlTranslatwn and Analysis of the Protein Products-The technology for production of capped RNAs by transcription of cloned genes under control of the bacteriophage SP6 promoter has been described previously (19,21). Translation of about 100 ng of RNA from each of the dhfr mutants was performed in reticulocyte lysates purchased from Bethesda Research Laboratories and in wheat germ extracts produced by the method of Anderson et

The abbreviations used are: dhfr, the mouse dihydrofolate reductase cDNA sequence; DHFR, the dihydrofolate reductase protein; SV40; simian virus 40; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography.

5031

5032 Translation Initiation at Non-A UG Triplets in Mammalian Cells al. (22). The products of in vitro translation were radiolabeled with ["Slmethionine or with [35S]tRNA"'et prepared by the method of Stanley (23) and analyzed by electrophoresis in polyacrylamide gels containing SDS (24). The N-terminal specificity of [35S]tRNANet labeling was confirmed by comparing the peptide maps of the products of translation of the wild-type dhfr sequence labeled with ["Slmethi- onine or ["S]tRNAY. The labeled proteins were subjected to electrophoresis in SDS-polyacrylamide gels and localized by autoradiography of the dried gel. Digestion with trypsin and elution of peptides from the gel were performed as described by Elder et al. (25). The tryptic peptides were applied to a reverse phase (Cl8) column for HPLC analysis and eluted in 0.1% trifluoroacetic acid with a 0-47.5% gradient of acetonitrile. Fractions were analyzed for the presence of "S by scintillation counting.

The Use of Viral Recombinants for Expression of the Mutant dhfr Sequences in Vivo-In order to ensure the expression of dhfr-specific mRNA at high levels in vivo, lytic SV40 viral recombinants were employed. Methods for the construction and propagation of SV40 recombinants containing the dhfr cDNA sequence in the late region have been described previously (20). The recombinants were constructed as plasmids in E. coli. Later the viral sequences were excised by restriction enzyme digestion, recircularized by ligation, and trans- fected (26) into CV1 cells in the presence of DNA from the SV40 early mutant, tsA58, as helper (27). High titer stocks of each recom- binant virus were produced and were used to infect CV1 cells a t multiplicities of infection of 1-10 plaque-forming units/cell. The presence of DHFR in cells infected with each of the recombinants was assessed by the Western blot method essentially as described by Burnette (28). dhfr-specific mRNA levels were measured by a slot- blot method (29) using as probe a purified restriction fragment containing the dhfr sequence, labeled with 32P by nick translation (30).

RESULTS

Utilization of Non-AUG Triplets in Vitro-The sequences of the wild-type and mutant translation initiation sites are shown in Fig. 1. Each of the 10 dhfr constructs in pSP64 (wild type and nine mutants) was digested to completion with EcoRI, which cuts the plasmid 3' to the dhfr sequence. This DNA was used as template in an in vitro run-off transcription reaction with SP6 RNA polymerase in the presence of the four nucleoside triphosphates and 'mGpppG. Under the con- ditions used the cap structure is efficiently incorporated at the 5' end of the transcript (21). The resulting RNAs were translated in wheat germ extracts or rabbit reticulocyte lysates, and the [35S]methionine-labeled products were fraction-

consensus ~ C C A U G G

dhf r wt AUCLAUG,G

m 2 ACG m21 AAG - m17 GUG - m22 AUA - m18 UUG - m23 AUC

m19 CUG - m 2 4 AULJ

m20 AGG - FIG. 1. The nucleotide sequences of the translation initia-

tion regions of the dhfr mutants used in this study. Each is the result of a single nucleotide substitution in the initiator AUG of dhfr. For comparison the eucaryotic translation initiation consensus sequence is also shown. wt, wild type.

ated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Fig. 2 (top panel) shows the products synthesized from the various mutant forms of dhfr mRNA in reticulocyte lysates. All of the mutants produce full-length DHFR but in markedly different quantities. In addition, each directs the synthesis of shorter forms of the protein. We cannot definitely identify these proteins, but the most abun- dant of them probably results from initiation at the next AUG triplet at codon 15. Like the normal initiation codon, this AUG resides within a favorable sequence context for translation initiation as defined by Kozak (15-17). The less abun- dant truncated species may result from initiation at a GUG triplet at codon 11. The amounts of full-length and truncated forms of DHFR were determined by scanning each lane of the autoradiogram with a densitometer. The quantities of full- length DHFR are expressed in Table I as the fraction of total protein synthesized. Of the non-AUG triplets tested, ACG and CUG are most efficiently recognized by the reticulocyte translation initiation apparatus. AAG and AGG, those triplets which contain a purine residue in the middle position, are least efficient. These results were confirmed by translation in wheat germ extracts as shown in Fig. 2 (lower panel) and summarized in numerical form in Table I. It is immediately apparent that the wheat germ system is less capable of initiation at non-AUG triplets than is the reticulocyte lysate.

h

FIG. 2. SDS-polyacrylamide gel electrophoresis of the products of translation of the wild-type and mutant forms of the dhfr sequence. The top p a n e l shows the results of translation in reticulocyte lysates and the lower p a n e l is from wheat germ extracts.

TABLE I Relative efficiencies of the various non-AUC triplets tested

in this study Each value represents the percent of total synthesis in full-length

DHFR Full-length DHFR

Reticulocyte Wheat germ Mutant

Wild type m2 (ACG) m17 (GUG) m18 (UUG) m19 (CUG) m20 (AGG) m21 (AAG) m22 (AUA) m23 (AUC) m24 (AUU)

?6 100 100 84 45 36 8 39 10 82 36 17" 3" 14" 3" 59 30 47 17 67 14

Probably do not initiate at the indicated triplets (see text).

Translation Initiation at Non-A UG Triplets in Mammalian Cells 5033

However, the relative initiation efficiencies of the various non-AUG triplets are similar in both systems. As before, ACG and CUG are the most efficiently utilized while AAG and AGG are recognized poorly, if at all. The other non-AUG triplets are utilized with intermediate efficiencies. Surpris- ingly, AUU, AUC, and AUA are utilized more efficiently than GUG and UUG, the most common bacterial alternatives to AUG.

It should be pointed out that the levels of initiation at AGG and AAG indicated in Table I are probably overestimates. In fact, little or no initiation apparently occurs with AGG and AAG. A close inspection of Fig. 2 indicates that the full-length products of translation of dhfr m20 (AGG) and m21 (AAG) do not exactly co-migrate with authentic DHFR. Their mobilities are slightly reduced. All the other non-AUG triplets direct the synthesis of products whose mobilities cannot be distinguished from wild type. This is more clear when they are separated on a longer (20 cm) gel (Fig. 3). The larger products of the AAG and AGG mutants may result from initiation at the non-AUG triplet (AUC) adjacent to the normal initiation codon (see Fig. 1). In any case, it is clear that the level of initiation at AAG and AGG is substantially lower than with the other triplets tested.

Utilization of the Non-AUG Triplets in Vivo-The ability of the various triplets to direct translation initiation in uiuo was determined by measuring the level of DHFR expressed from SV40 recombinants containing the mutant sequences. Their construction and properties have been described in previous reports (11,20) and under "Materials and Methods." Monolayers of CV1 cells were infected with each of the recombinants at multiplicities of infection of 1-10 plaque- forming units/cell. Protein and RNA were extracted at late times of infection. The amounts of dhfr-specific mRNA produced in each infection were quantitated by a slot-blot method (29) and used to determine how much of each protein extract to apply to the SDS-polyacrylamide gel. In order to clearly resolve the products of the transduced mouse dhfr sequences from endogenous monkey DHFR, it was necessary to use a gel about 40 cm in length. After fractionation by electrophoresis the proteins were transferred to a nitrocellulose mem- brane and DHFR was visualized using antibody against DHFR and '251-protein A. Fig. 4 shows that the results of this experiment approximately parallel those from the in vitro translations. As observed previously ( l l ) , the non-AUG triplets appear to be much less efficiently recognized in vivo than in vitro, but the overall hierarchy of efficiencies is similar. ACG was the most efficient, followed by CUG, AUC,

IVI"

FIG. 3. The products of translation of the mutant dhfr sequences in reticulocyte lysates separated on a 20-cm SDS- polyacrylamide gel.

FIG. 4. Western blot analysis of DHFR produced in uiuo from CVl cells infected with SV40 recombinants containing the wild-type and mutant dhfr sequences. The upper band of the doublet is endogenous monkey cell DHFR, and the lower band represents the product of the heterologous mouse sequence. DHFR species were visualized by autoradiography of the nitrocellulose mem- brane after treatment with antibody to the mouse enzyme and radi- oiodinated protein A.

8000

7000 1 A I I

6000

5000

4000

3000

2000

1000

n " 10 20 30 40 50 60 70 80 90

FRACTION NO.

2ooo I B

looot 0

10 20 30 40 50 60 70 80 90

FRACTION NO.

FIG. 5. A, the HPLC elution profile of tryptic peptides of DHFR produced in vitro in the presence of [%]methionine. B, peptides labeled by translation in the presence of [35S]tRNA?".

AUU, and AUA. No full-length DHFR was detected when GUG, UUG, AAG, or AGG was the initiator triplet. No species is present which might correspond to initiation a t internal positions as was seen in vitro (Figs. 2 and 3). Perhaps this truncated form of DHFR is rapidly degraded in uiuo. We cannot exclude the alternative possibility that the AUG at codon 15 is not recognized as an initiation site by ribosomes in vivo.

Translation Initiation at Non-A UG Triplets Utilizes Methi- onine-In vitro translation in the presence of [35S]tRNAp is frequently used to study translation initiation. Since the radioactive amino acid is capable of being incorporated only at the amino terminus, labeling of the protein indicates initiation with methionine. We prepared [35S]tRNAp by the method of Stanley (23) and added it to in vitro translation reactions. To confirm that this material specifically labeled the amino terminus of DHFR the products of translation of

5034 Translation Initiation at Non-A UG Triplets in Mammalian Cells

FIG. 6. SDS-polyacrylamide gel electrophoresis of the in vitro translation products of the wild-type and mutant dhfr sequences labeled with [35S]tRNAf"". The upper panel is from reticulocyte lysates, and the lower panel is from wheat germ extracts.

the wild-type dhfr sequence were subjected to tryptic peptide analysis as described under "Materials and Methods." [35S] Methionine labeled many peptides, but [35S]tRNAy labeled only one (see Fig. 5), consistent with the assertion that only the amino terminus became labeled.

The various forms of dhfr mRNA were translated in vitro using both the wheat germ and reticulocyte lysate systems, and the products were fractionated by SDS-polyacrylamide gel electrophoresis. The results of this analysis are shown in Fig. 6. The pattern of labeled proteins is very similar to that observed when the protein is labeled at internal positions using [35S]methionine (compare to Fig. 2), indicating that in each case the polypeptide is initiated with methionine. Al- though it seems clear that methionine is utilized to initiate translation of the mutant sequences, we cannot categorically rule out the possibility that some fraction of the polypeptide chains is initiated with another amino acid. Amino acid sequence analysis of the product of translation of the ACG mutant suggests, however, that little or no utilization of an amino acid other than methionine occurs (11).

DISCUSSION

The ability of mammalian ribosomes to initiate translation at triplets other than AUG emphasizes the complexity of eucaryotic translation initiation sites and adds to the clear and growing evidence that sequences in the vicinity of an initiation codon contribute to its ability to function in translation initiation. Since non-AUG triplets are normally ignored by the translation initiation apparatus, the occurrence of initiation events at such sites indicates that sequences outside the initiation codon must play a role. This expectation has been borne out experimentally. The initiator consensus sequence originally defined by Kozak (11, 15-17) is clearly an important determinant of the efficiency with which AUG and non-AUG triplets are recognized as initiation codons.

Since initiation at a non-AUG triplet requires base mis- match at the level of the codon-anticodon interaction, the efficiency with which some non-AUG triplets are recognized as initiation codons is a little surprising. In uiuo ACG is utilized with as much as 10-15% the efficiency of AUG in some experiments. This is an error frequency that would be intolerable during the elongation phase of protein synthesis. Since some base mismatches seem to be tolerated remarkably well, it is clear that the interaction between the codon and anticodon cannot be an over-riding determinant of the fidelity

of initiation codon recognition. As mentioned above, sequence context helps define an initiation site. Moreover, a high degree of fidelity is apparently introduced into the initiation process at the level of ternary complex formation (eukaryotic initiation factor 2-GTP-tRNAMet). Methionine is the initiating amino acid regardless of which triplet is being utilized, because tRNAF is probably the only species able to gain access to the initiation site. These considerations allow the require- ments for precise codon-anticodon interaction to be less strin- gent than those which operate during elongation.

The different efficiencies of utilization of the various non- AUG triplets suggest that some nucleotide mismatches are less destabilizing than others. It has been recognized for many years that codon-anticodon interactions can involve unconventional base pairing schemes that render certain mismatches tolerable. This was the basis of Crick's wobble hy- pothesis which predicted alternative base pairing schemes for the third position of some codons (31). Other unusual base pairing schemes have also been discussed relative to the fidelity of codon-anticodon interaction (32). The possibility of additional unconventional base pairs has been inferred from studies of natural suppressor tRNAs (33), from analysis of the secondary and tertiary structures of ribosomal RNAs (34) and tRNAs (35, 36), and from the three-dimensional structures of mismatched synthetic duplex oligodeoxyribonucleotides (37-39). Some of these unconventional base pairs may form during initiation at the non-AUG triplets examined in this study. Unfortunately, the current state of knowledge of the destabilizing effects of specific single base mismatches in RNA seems insufficient to permit one to conclude whether the strength of the codon-anticodon interaction is the sole determinant of the relative initiation efficiencies of the non- AUG triplets. Moreover, conformational constraints imposed on the anticodon by the secondary structure of tRNA, and by the environment of the ribosome itself, may make it difficult to extrapolate directly from studies of model oligonucleotides. However, we can point out that some positions of the initiator triplet are more sensitive to substitution than others. Thus, the third position is relatively insensitive to substitution, each of the mutations being tolerated about equally well. The first position can also be altered without eliminating the ability to serve as an initiator, although C is clearly favored over U or G. The second position is more sensitive to substitution and cannot tolerate the presence of a purine residue, even though a C at this site yields the most efficient of all the non-AUG initiation codons tested in this study.

There are at least two potential advantages in the ability to initiate translation at sites other than AUG. 1) Since initiation at a non-AUG triplet is generally less efficient than at AUG, it could provide a means for negatively modulating the synthesis of a protein that is required (or tolerated) only in small quantities. 2) Initiation at a non-AUG triplet situated upstream of an initiator AUG provides a mechanism for the production of more than one protein from a single mRNA. In some instances this has been accomplished by altering the sequence context of an AUG to reduce its efficiency and thus reveal the presence of a downstream initiation codon. The synthesis of virion proteins 2 and 3 of SV40 is an example of such a mechanism. However, the same goal can be accomplished by the use of a non-AUG initiator triplet.

So far only a few examples of translation initiation at naturally occurring non-AUG triplets have been reported. Adeno-associated virus initiates translation of a capsid protein with ACG (7). An ACG is apparently also used as an initiation codon for the C' protein of Sendai virus (8, 9). Moreover, recent evidence indicates that the c-myc gene di-

Translation Initiation at Non-AUG Triplets in Mammalian Cells 5035

rects the synthesis of an alternative product by initiation at a CUG triplet upstream of the initiator AUG (12). These results emphasize the possibility that additional examples of this phenomenon will yet be found.

Acknowledgments-We thank Gavin Pickett for technical assist- ance, Latif Kazim for help with HPLC, and Elvira Ehrenfeld for advice on the fMet labeling experiments.

REFERENCES

1. Gren, E. J. (1984) Biochimie (Paris) 66, 1-29 2. Kozak, M. (1983) Microbiol. Rev. 47, 1-45 3. Clark, B. F. C., and Narcker, K. A. (1966) J. Mol. Bwl. 17,394-

4. Van der Laken, K., Bakker-Steenveld, H., and Van Knippenberg,

5. Van der Laken, K., Bakker-Steenveld, H., Berkhout, B., and Van Knippenberg, P. H. (1980) Eur. J. Biochem. 104.19-23

6. Sherman, F., McKnight, G., and Stewart, J. W. (1980) Biochim. Biophys. Acta 609,343-346

7. Becerra, S. P., Rose, J . A,, Hardy, M., Baroudy, B. M., and Anderson, C. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 76,

406

P. (1979) FEBS Lett. 100, 230-234

7919-7923 8. Curran, J., and Kolakofsky, D. (1988) EMBO J. 7, 245-251 9. Gupta, K. C., and Patwardhan, S. (1988) J. Biol. Chem. 263,

10. Anderson, C. W., and Buzash-Pollert, E. (1985) Mol. Cell. Biol.

11. Peabody, D. S. (1987) J. Biol. Chem. 262, 11847-11851 12. Hann, S. R., King, M. W., Bentley, D. L., Anderson, C. W., and

13. Clements, J. M., Laz, T. M., and Sherman, F. (1988) Mol. Cell.

14. Zitomer, R. S., Walthall, D. A., Rymond, B. C., and Hollenberg,

15. Kozak, M. (1984) Nature 308, 241-246

8553-8556

6,3621-3624

Eisenman, R. N. (1987) Cell 62,185-195

Biol. 8,4533-4536

C. P. (1984) Mol. Cell. Biol. 4, 1191-1197

16. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872 17. Kozak, M. (1986) Cell 44,283-292 18. Sanger, F., Nicklen, and Coulson, A. R. (1977) Proc. Natl. Acad.

Sci. U. S. A. 74, 5463-5467 19. Melton, D. A., Krieg, P., Rebagliati, M. R., Maniatis, T., Zinn,

K., and Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7056 20. Subramani, S., Mulligan, R. C., and Berg, P. (1981) Mol. Cell.

Biol. 1,854-864 21. Pelletier, J., and Sonenberg, N. (1985) Cell 40, 515-526 22. Anderson, C. W., Straus, J. W., andDudock, B. S. (1983) Methods

23. Stanley, W. M. (1974) Methods Enzymol. 29, 530-565 24. Laemmli, U. K. (1970) Nature 227,680-685 25. Elder, J. H., Pickett, R. A., 11, Hampton, J., and Lerner, R. A.

(1977) J. Bwl. Chem. 262,6510-6515 26. Graham, F. L., and Van der Eb, A. J. (1973) Virology 62, 456-

467 27. Mertz, J. E., and Berg, P. (1974) Virology 62, 112-124 28. Burnette, W. N. (1981) Nucleic Acids Res. 112, 195-203 29. Meinkoth, J., and Wahl, G. (1984) Anal. Biochem. 138, 267-284 30. Rigby, P. W., Dieckmann, M., Rhodes, C., and Berg, P. (1977) J.

31. Crick, F. H. C. (1966) J. Mol. Biol. 19, 548-555 32. Topal, M. D., and Fresco, J. R. (1976) Nature 263,289-293 33. Valle, R. P. C., Morch, M.-D., and Haenni, A.-L. (1987) EMBO

34. Noller, H. (1984) Annu. Reu. Biochem. 63, 119-162 35. Rich, A., and RajBhandary, U. L. (1976) Annu. Reu. Biochem.

36. Jack, A., Ladner, J. E., and Klug, A. (1976) J. Mol. Biol. 108, 619

37. Brown, T., Hunter, W. N., Kneale, G., and Kennard, 0. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2402-2406

38. Hunter, W. N., Brown, T., Anand, N. N., and Kennard, 0. (1986) Nature 320,552-555

39. Prive, G. G., Heinemann, U., Chandrasegaran, S., Kan, L.-S., Kopka, M. L., and Dickerson, R. E. (1987) Science 238, 498- 504

Enzymol. 10 1,635-644

Mol. Biol. 113, 237-251

J. 6,3049-3055

46,805-860

translation initiation at non-aug triplets in mammalian cells* · expression in cultured monkey...

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