Mar 24, 1986 - uridine in position 36 is also required but not for base paiIng. A number of cases involving viruses, transposons, and cellular genes have been ...
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 5062-5066, July 1986 Biochemistry
tRNA anticodon replacement experiments show that ribosomal frameshifting can be caused by doublet decoding (T4 RNA ligase/celi-free protein synthesis)
A. G. BRUCE*t, J. F. ATKINS*t,
AND
R. F. GESTELAND*
*Howard Hughes Medical Institute, Departments of Biology and Human Genetics, University of Utah, Salt Lake City, UT 84132; and tDepartment of Biochemistry, University College Cork, Cork, Ireland
Communicated by Sidney Velick, March 24, 1986
reading either its conventional triplet codon out of frame or a related codon in a nontriplet manner. Normal tRNAs can cause frameshifting in in vitro protein synthesis experiments using extracts from E. coli (11). Normal tRNAs are also likely to be the cause of the frameshifting detected in in vivo experiments with aminoacylation limitation (12) and in studies of frameshift mutant leakiness (13, 14). In our in vitro experiments with bacteriophage MS2 RNA as mRNA, 36 tRNAs were tested (11). Only two, tRNAser and tRNA3hr, were able to produce high levels of frameshifting near the end of the coat protein gene. The AGE decoding tRNA~er stimulates the synthesis of two proteins, called 6 and 7, that are longer than the coat protein (Fig. 1). Amino acid sequencing of the proteins shows that the frameshifting occurs at any one of four GCA alanine codons due to apparent doublet decoding (16). Frameshifting at the first of the four alanine codons gives protein 6, while protein 7 is a mixture of three proteins from shifting at the other three alanine codons. (Proteins 6 and 7 have the same number of amino acid residues but are separable by gel electrophoresis, probably due to the difference in composition.) The mechanism for this frameshifting is not obvious because the anticodon of tRNASer is not complementary to the alanine codon. The models that have been proposed involve conventional base pairing between only the first two bases of the codon (GC) and bases 35 and 34 (CG) of the anticodon but they differ as follows in the possible involvement of other base pairing (11, 17):
The expression of certain normal genes reABSTRACT quires a specific ribosomal frameshift event because the mRNA has the coding information for one protein in two different reading frames. One of several possible mechanisms for this involves recognition of a nontriplet codon by a noncognate tRNA. The AGE-decoding Escherschia coli tRNA1 reads a GCA alanine codon to cause a -1 fraresbift. Replacement of the anticodon of tRNA"t' with the anticodon of tRNAJF allows the constructed tRNA to cause this frameshifting. By altering the anticodon loop nucleotides at positions 33-36 in the constructed tRNI~e molecules, the tRNA was found to recognize a 2-base codon. Instead of the usual anticodon, positions 34-36, the nucleotides in positions 34 and 35 form essential base pairs with the first two positions of the alanine codon. The uridine in position 36 is also required but not for base paiIng. A number of cases involving viruses, transposons, and cellular genes have been described where the expression of a normal gene involves a rare but specific nontriplet decoding event. This results in the synthesis of a minor proportion of the protein having a different carboxyl terminus that is either elongated or truncated, compared to the major protein, depending on the distribution of termination codons in the new reading frame. This unusual mechanism may be used to control the expression of certain proteins that are only required in small amounts. For instance, the reverse transcriptase of Rous sarcoma virus is encoded by the pol gene, immediately downstream from the gag gene, and is expressed only as a gag-pol precursor protein. Only a small amount of the precursor is made relative to the gag product. However, the gag and pol genes are not in the same reading frame, and the precursor is made only if a ribosome shifts to the -1 frame prior to the gag gene termination codon (1). By inference from nucleotide sequences, a similar frameshift mechanism is likely to be used by several other retroviruses (1, 2) and by the yeast transposon Ty-1 (3, 4). The synthesis of Escherichia coli release factor 2 protein, which causes polypeptide chain termination at UAA and UGA codons, also requires a shift in reading frame (5). The gene has an in-frame UGA at codon 26 with an open reading frame continuing in the + 1 frame. The protein sequence shows that a frameshift event occurs immediately before the termination codon to permit ribosomes to bypass the terminator and synthesize the full-length protein. Ribosomal frameshifting is also involved in the production of two bacteriophage T7 proteins (6) and perhaps in an alfalfa mosaic virus protein (7) and certain proteins encoded in mitochondria (8-10). These naturally occurring frameshift events could involve an unusual tRNA that exclusively reads a nontriplet codon. Alternatively a normal tRNA could cause frameshifting by
Anticodon Codon
3' U C G U33 5' 5' G C (A) 3'.
This model differs from the "two-out-of-three" proposal of Lagerkvist (18) in that it involves normal pairing with positions 34 and 35 rather than 35 and 36. To determine the mechanism of this unusual codonanticodon interaction, we have constructed a series oftRNAs with altered anticodon loops. By determining the ability of these altered tRNAs to cause frameshifting in the in vitro protein synthesizing system, we have characterized the features of the tRNA that are important for this decoding process. MATERIALS AND METHODS Enzymes. T4 RNA ligase was purified from E. coli containing KR54, which overproduces the enzyme (19), using the method of Brennan et al. (20) through the DE-51 step, followed by Blue Sepharose (21). E. coli tNAPhC ligase (formerly synthetase) was purified from cells containing pIDi, which overproduces the enzyme 10-fold (22), using the method of Stulberg (23) except the hydroxyapatite column was replaced with Sephadex G-200. Yeast nucleotidyltransferase was purified as described by Sternbach et al. (24). T4 polynucleotide kinase was a gift from 0. Uhlenbeck. Poly-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
tPresent address: Oncogen, 3005 1st Avenue, Seattle, WA 98121. 5062
Proc. Natl. Acad. Sci. USA 83 (1986)
Biochemistry: Bruce et al. s
5063
+1 Frame
MS2
ynthetase 62K
3
COAT
-1 FRAME
ALA PRO ALA ALA STOP STOP --GUU AAG GCA--(9)--CCG--(2)--UCA GCA AUC GCA GCA--(4)--UAC UAA--(23)--AUU GAU--
I
I
I
GC GC
GC GC
-cuProte-n -CUA-A
-UGA
7
-CUA-A
-UGA
6
FIG. 1. Frameshift sites near the end of the MS2 coat protein gene (16) that give rise to elongated proteins due to a shift to the -1 reading frame. Numbers in parentheses indicate intervening codons. Termination codons are underlined, and reading frames are indicated by the grouping of triplets.
were desalted by lyophilization overnight. The identity of the oligonucleotide was confirmed as described (25). The 5' phosphate was added by incubation with ATP and polynucleotide kinase (27). Transfer RNA. E. coli tRNAser, tRNAThr, tRNAAl, and tRNALYs were from Subriden. tRNAPhe was isolated from E. coli containing pRK3, which overproduces tRNAPhC about 10-fold (28). The general method for the construction of the tRNAs (Fig. 2) will be described in detail elsewhere. The tRNAs were purified by urea gel electrophoresis and sequenced as described (25). In each case the tRNA was judged to be at least 95% the desired product. The most likely contaminant is joined half molecules with no oligonucleotide inserted. Such a contaminant does not cause frameshifting in the assay described below. The tRNAs (except tRNAS'C were aminoacylated with [3H]phenylalanine (29.1 Ci/mmol; 1 Ci = 37 GBq; Amersham) using purified E. coli tRNAPhe ligase in the presence of dimethyl sulfoxide (29). After phenol extraction and ethanol precipitation 12-23% was recovered in the aminoacyl form as determined from trichloroacetic acidprecipitable counts.
nucleotide phosphorylase (from Micrococcus lysodeikticus and E. coli) and ribonuclease A were purchased from Sigma. Ribonuclease T1 was from Calbiochem and bacterial alkaline phosphatase (BAPC) was from Cooper Biomedical (Malvern, PA). Oligoribonudeotides. GpGpC and GpGpU were isolated from a complete RNase A digestion of poly (G-C) and poly (G-U). The remaining trinucleotides were synthesized enzymatically using M. lysodeikticus polynucleotide phosphorylase (25). CpGpC, UpGpC, ApCpU, CpCpU, UpCpU, GpUpU, GpCpC, GpCpA, GpCpG, GpCpU, and GpApA were synthesized by the equilibrium method. GpApU and ApGpC were synthesized by the RNase A-assisted method. The tetranucleotides were synthesized by addition of a single nucleotide to the appropriate trinucleotide using T4 RNA ligase (26). GpC was purchased from Sigma. The terminal phosphates were removed by incubation with alkaline phosphatase. The oligonucleotides were purified by HPLC on a 4.5 x 300 mm Zorbax-NH2 column using a linear gradient from 20 to 300 mM ammonium acetate, pH 4.5, where they elute primarily according to length. The oligonucleotides AOH
C AOH
2
c A-
%
c pGC
, L_
*,1) Si nuclease 2) P.A.GE.
; A\ A* U --G A A "'-
2
AOH
c Cp/
COH
C
A
A
A
pGC
pGC
pGC
_2
L
~ U
A
A*
> Kinase-phosphatase
NNucleotidyl transferase
guanosine > cytidine). Position 34. The results of substitutions for the guanosine at position 34 are shown in lanes 10-12. Alteration of guanosine-34 to adenosine greatly reduces activity and minimal activity is seen with cytidine or uridine (i.e., guanosine >> cytidine > uridine > adenosine). Position 35. Lanes 13 and 14 show that substituting cytidine-35 with adenosine significantly reduces frameshifting and that uridine shows little, if any, activity (i.e., cytidine >> adenosine > uridine). With guanosine-35 (lane 15) the tRNA stimulates synthesis of the frameshift protein 7, but not protein 6. This tRNA now has an anticodon sequence 5' GGU 3' that is the same as that in tRNAphr, the only other normal tRNA shown to give a high level of frameshifting. tRNAphr stimulates synthesis of protein 7, but not protein 6 (11), and amino acid sequencing (16) has shown tRNAThr causes a -1 frameshift event that occurs at the proline codon shown in Fig. 1. However, position 35 must be a cytidine for the constructed tRNA to give the same frameshift products as tRNASer. This is a striking result because it again shows the importance of the anticodon in determining whether the tRNA promotes frameshifting. Position 36. If the uridine at position 36 is substituted with any of the other three bases, little if any stimulation of frameshifting is observed. We conclude that to obtain efficient frameshifting of this type it is necessary to maintain the tRNAser anticodon, but there is greater latitude in the identity of base 33 than for base 34, 35, or 36. It is necessary to consider the possibility that the constructed tRNAs that did not stimulate synthesis of the
RESULTS The experiments reported here were performed by excising a part of the anticodon loop of a tRNA and replacing it with an oligoribonucleotide of choice. The large variable loop of tRNA~er makes it difficult to find cleavages specific for the anticodon. Instead a method was developed for altering the anticodon loop of E. colitRNAPhe. The anticodon, 5' GAA 3', was specifically excised with S1 nuclease and was replaced with a trinucleotide of desired sequence using T4 RNA ligase. Using a modification of this procedure the tetranucleotide UGAA was removed to allow alteration of nucleotide 33 as well as the anticodon (positions 34, 35, and 36). The anticodon region of yeast RNAPhe is a recognition site for an aminoacyl-tRNA ligase (25). Similarly a constructed E. coli tRNAPhe with a GCU anticodon was aminoacylated poorly by an E. coli S-100 extract. To ensure that any observed differences in activity of the constructed tRNAs were not due to differences in their ability to be aminoacylated, each tRNA was aminoacylated with phenylalanine using conditions to relax the enzyme specificity (29). This was essential because if the tRNAs were not aminoacylated, only the tRNA with 5' GGU 3' inserted was able to cause significant levels of frameshifting in the assay described below. The ability of tRNAs to stimulate frameshifting, as monitored by synthesis of the elongated MS2 coat proteins 6 and 7, is shown in Fig. 3. The background level of frameshifting is shown in lane 2. Addition of tRNAPhe (lane 3) or a constructed tRNA with the normal phenylalanine anticodon 5' GAA 3' inserted (lane 4) shows no stimulation. tRNAser has the anticodon 5' GCU 3'. When this anticodon replaces the normal anticodon of tRNAPhe, the resulting tRNA, lanes 9 and 19, is at least as active as tRNA~sr, lane 5, in stimulating synthesis of MS2 frameshift proteins 6 and 7. The predominant effect is on protein 7, presumably reflecting the fact that there are three sites for frameshifting to give protein 7 but only one for protein 6 (Fig. 1). It was shown before that frameshifting stimulated by tRNASer is diminished by addi1
2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18 192021 -
me"
____0__4_____
^= 4 k
sw ill
=
t wb " ^
;
A
5 -._ 6-
5
-6
7-
7 a-
mRNA tRNA
-
33 34 35 36
66K
_*.synth.
-Phe
Ser
GAC u G A
A
G
G G G G ACUG G G G G G G G C CC CC CC C UAGc cc cc u
uuuuuuuuuu
CAG u - -
COAT
FIG. 3. Effect of altering the anticodon loop nucleotides on the ability of constructed tRNAs to promote frameshifting. The oligonucleotides inserted into E. coli tRNAPhe in place of nucleotides 33-36 (lanes 6-9 and 21) or nucleotides 34-36 (lanes 4 and 10-20) are shown. The bold letters indicate differences from the E. coli tRNA3ser. An autoradiograph of in vitro protein synthesis products electrophoresed on a 15% polyacrylamide/NaDodSO4 gel is shown.
Proc. Natl. Acad. Sci. USA 83 (1986)
Biochemistry: Bruce et al.
U
G *C C
S4 G
GA
CG G
GG
DG AA
A
G
G
C.*GU * A U *A
U
C'*G
G
G.*U
C G G
.0 GG*G
s2c
U A C AA
A U At6 GC U
C G
pG *'C C *G
*G *G *G *C *C *U U GAGCC A
4t uA
U A
C C A
A C
pG C C C G G
. . . CUC C U AGGOm G * C x A GDA G 7m G' CG U G* C G C
U
GAA
R
AOH
AOH C C
Aom C C G pG C G *C U A G C A
tRNAT
tRNAPHE
tRNAS R
5065
.GT
G
A U
*.U *CG A~
DGA A ... ...G DCUC .G GGC A G T ,IC G GAGOC U GDA G G7m C * G U A U Amt6 GGU
A ms2i6
FIG. 4. Comparison of the primary structures of E. coli tRNAs. tRNA3er (34) and tRNAP1 (35) cause significant levels of frameshifting in the translation of the MS2 coat gene while the tRNAl'e (36) does not. Substituting the anticodon of either the tRNA3er or tRNA3 r for the GAA of tRNA le causes this tRNA to become shifty.
frameshift proteins 6 and 7 are defective for protein synthesis altogether, although there is no obvious reason to think so. The tRNAs were all aminoacylated with phenylalanine to avoid problems with ligase recognition. In certain cases we can infer that these tRNAs are functional. In Fig. 3, lanes 13 and 18, the mobility of coat protein has increased. These tRNAs have anticodons complementary to arginine and lysine codons, and it seems reasonable to conclude the insertion of phenylalanine at these sites has increased the net negative charge and thus increased the electrophoretic mobility. [The opposite result has been observed when a large excess of tRNALYs is added to the in vitro protein synthesizing system (data not shown).] The tRNA with the anticodon 5' GCC 3' stimulates synthesis of a protein ofabout 67 kDa that comes from the MS2 synthetase gene by some unknown mechanism.
between the two tRNAs (Fig. 4) strikingly demonstrate that it is the anticodon region of the tRNA that is most important in determining if the tRNA will be shifty. Fig. 5 shows a proposal for the codon-anticodon interaction involved in this frameshifting. The conventional recognition of the AGC (serine) codon by tRNA3er is shown in Fig. 5A. Fig. 5B is a model for how this tRNA could recognize the GCA alanine codon to cause -1 frameshifting. Fig. SC shows this same interaction but with the constructed anticodon loop. The anticodon is reduced to the two nucleotides that would normally recognize nucleotides 2 and 3 of the seine codon now pairing with nucleotides 1 and 2 of the alanine codon. The results show that the guanosine at position 34 and the cytidine at position 35 are essential. Uridine at position 36 is also important, but not due to involvement in base pairing. If it formed a base pair, it would be with the third nucleotide of the codon previous to the alanine codon. For the four sites where frameshifts are known to occur this is guanosine, adenosine, or cytidine (Fig. 1). The normal tRNASer with uridine-36 could not pair with the cytidine by conventional rules, yet frameshifting is observed at this site (16). Also as shown here, with cytidine-36 frameshifting was not detected at any of these sites even though it could have paired with the upstream guanosine of one of them. The other known tRNAs
DISCUSSION Features of tRNA~sr that are important for frameshifting have been defined. Inserting the anticodon of the "shifty" tRNAser (or tRNATh ) into a "nonshifty" tRNAPhe produces a tRNA that is as efficient as the parent tRNA at causing frameshifting. This result and the major sequence differences
A
C
B
reconstructed tRNASER
3'
G * C A * U G * C G * C G * C
A
tRNAS3R
5'
G A G G G
S2 C
t6A
3'
U
36 35 34
A t6 A36
zero frame
SER
5'
S2C 33 35 34 *
5'A G C
C U C C 0C
3'
ALA
C C C C
e G
5'
* G * G * G ;, . A A 33
ms2i6A 36
C G
S
5G C
3'
35 34
C G
U C G
mRNA
* * * *
tRNAPHE
*
(A)3'
-1 framee
S
5 G C
(A)3
ALA
FIG. 5. (A) Binding of tRNASer to the normal serine codon AGC. (B) Proposed binding of tRNA1er to the alanine codon GCA. (C) Binding of a constructed tRNA with 5' UGCU 3' inserted in the anticodon loop binding to the alanine codon. This tRNA has the same anticodon as
tRNA3Ser.
5066
Biochemistry: Bruce et al.
that have guanosine-34 and cytidine-35 and hence might give frameshifting at GCA alanine codons by this mechanism have the anticodons 5' GCA 3' (tRNACYS), GCC (tRNAGly), and the anticodon ICG (according to wobble rules inosine pairs with cytidine, uridine, or adenosine) occurs in tRNA'9 (37). These tRNAs do not have uridine at 36 and do not stimulate synthesis of proteins 6 or 7. The other known tRNAs that are capable of forming two G-C base pairs also do not have uridine at position 36. Several of these tRNAs have been shown to be unable to promote significant levels of frameshifting (11). These results suggest that having uridine at position 36 is essential for frameshifting but not for reasons of base pairing. Possibly it allows the anticodon loop to adopt an unusual conformation that permits this stable 2-base codon-anticodon interaction. It might be expected that this 2-base codon recognition would be enhanced in a tRNA that has a 6-member anticodon loop rather than the normal 7. However, if uridine-36 or -33 are deleted, the ability to cause frameshifting is lost (Fig. 3, lanes 20 and 21). The smaller loops of these tRNAs are probably unable to adopt the conformation that is required for recognition and/or translocation. tRNAPhe molecules with a GCU anticodon and with position 33 being adenosine or uridine are equally able to stimulate the synthesis of the frameshift proteins 6 and 7. If the interaction between uridine-33 and the adenosine of the GCA codon (Fig. 5) were required for stabilization, the tRNA with adenosine-33 should be less effective at causing frameshifting. The occurrence of A-A pairing would not strengthen the decoding interaction. This clearly demonstrates that the proposed formation of a transient base pair between the nucleotide at position 33 and the third base of the codon (17) is not required. Nor does a base pair involving uridine-33 and the flanking 3' codon nucleotide appear to be the explanation for the context effect in nonsense suppression (33). In a study of tRNAs with 7-membered (38) or 8-membered (15) anticodon loops, the potential importance of uridine at position 33 and/or 34 in permitting a bend in the anticodon loop important for codon binding was discussed. The present study shows that a bend due to uridine-33 is not required for this type of doublet decoding. The position 37 nucleotide on the 3' side of the anticodon of tRNAser is the hydrophilic N6-(N-threonylcarbonyl)-2'-Omethyl-adenosine (t6A) but in the constructed tRNAs it is the very different hydrophobic N6-isopentenyl-2-methylthioadenosine (ms2i6A), yet the tRNAs are equally efficient at causing frameshifting. Therefore, the identity of this modification does not play an important role. From these experiments and the amino acid sequencing of the frameshift proteins (16), we conclude that tRNAser causes a frameshift at alanine codons by a 2-base interaction involving conventional base pairs between positions 34 and 35 of the tRNA and the first two bases of the codon. In other cases where frameshifting is known to occur, doublet decoding by the formation of two GC base pairs is not necessarily involved. It is likely that other mechanisms of frameshifting exist. Each of the different mechanisms will need to be characterized to predict the sites of frameshifting and to understand the potential regulatory functions. We thank Marie Havlick for excellent technical assistance. This work was supported by Grant DMB-8408649 from the National Science Foundation to R.F.G. and J.F.A., Grant 18/82 from the Irish National Board for Science and Technology to J.F.A., and by the Howard Hughes Medical Institute. 1.
Jacks, T. & Varmus, H. E. (1985) Science 230, 1237-1242.
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82, 2829-2833. 4. Mellor, J., Fulton, S. M., Dobson, M. J., Wilson, W., Kingsman, S. M. & Kingsman, A. J. (1985) Nature (London) 313, 243-246. 5. Craigen, W. J., Cook, R. G., Tate, W. P. & Caskey, C. T. (1985) Proc. Natl. Acad. Sci. USA 82, 3616-3620. 6. Dunn, J. J. & Studier, F. W. (1983) J. Mol. Biol. 166, 477-535. 7. Joshi, S., Neeleman, L., Pleij, C. W. A., Haenni, A. L., Chapeville, F., Bosch, L. & Van Vloten-Doting, L. (1984) Virology 139, 231-242. 8. Hensgens, L. A. M., Brakenhoff, J., De Vries, B. F., Sloof, P., Tromp, M. C., Van Boom, J. H. & Benne, R. (1984) Nucleic Acids Res. 12, 7327-7344. 9. de la Cruz, V. F., Neckelmann, N. & Simpson, L. (1984) J. Biol. Chem. 259, 15136-15147. 10. Michael, N. L., Rothbard, J. B., Shiurba, R. A., Linke, H. K., Schoolnik, G. K. & Clayton, D. A. (1984) EMBO J. 3, 3165-3175. 11. Atkins, J. F., Gesteland, R. F., Reid, B. R. & Anderson, C. W. (1979) Cell 18, 1119-1131. 12. Weiss, R. & Gallant, J. (1983) Nature (London) 302, 389-393. 13. Atkins, J. F., Nichols, B.P. & Thompson, S. (1983) EMBO J. 2, 1345-1350. 14. Weiss-Brummer, B., Huttenhofer, A. & Kaudewitz, F. (1984) Mol. Gen. Genet. 198, 62-68. 15. Bossi, L. & Smith, D. M. (1984) Proc. Natl. Acad. Sci. USA 81, 6105-6109. 16. Dayhuff, T. J., Atkins, J. F. & Gesteland, R. F. (1986) J. Biol. Chem., in press. 17. Weiss, R. B. (1984) Proc. Natl. Acad. Sci. USA 81, 5797-5801. 18. Lagerkvist, U. (1978) Proc. Natl. Acad. Sci. USA 75, 17591762. 19. Rand, K. N. & Gait, M. J. (1984) EMBO J. 3, 397-402. 20. Brennan, C. A., Manthey, A. E. & Gumport, R. I. (1983) Methods Enzymol. 100, 38-52. 21. Sugiura, M. (1980) Anal. Biochem. 108, 227-229. 22. Elseviers, D., Gallagher, P., Hoffman, A., Weinberg, B. & Schwartz, I. (1982) J. Bacteriol. 152, 357-362. 23. Stulberg, M. P. (1967) J. Biol. Chem. 242, 1060-1064. 24. Sternbach, H., von der Haar, F., Schlimme, E., Gaertner, E. & Cramer, F. (1971) Eur. J. Biochem. 22, 166-172. 25. Bruce, A. G. & Uhlenbeck, 0. C. (1982) Biochemistry 21,
3921-3926. 26. Bruce, A. G., Atkins, J. F., Wills, N., Uhlenbeck, 0. C. & Gesteland, R. F. (1982) Proc. Natl. Acad. Sci. USA 79, 71277131. 27. Uhlenbeck, 0. C. & Cameron, V. (1977) Nucleic Acids Res. 4, 85-98. 28. Schwartz, I., Klotsky, R. A., Elseviers, D., Gallagher, P. J., Krauskopf, M., Siddiqui, M. A. Q., Wong, J. F. H. & Roe, B. A. (1983) Nucleic Acids Res. 11, 4379-4389. 29. Giege, R., Kern, D. & Ebel, J. P. (1972) Biochimie 54, 1245-1255. 30. Bare, L., Bruce, A. G., Gesteland, R. F. & Uhlenbeck, 0. C. (1983) Nature (London) 305, 554-556. 31. Thompson, R. C., Cline, S. W. & Yarus, M. (1982) in Interaction of Translational and Transcriptional Controls in the Regulation of Gene Expression, eds. Grunberg-Monago, M. & Safer, B. (Elsevier, New York), pp. 189-202. 32. Bruce, A. G. & Gesteland, R. F. (1982) in Interaction of Translational and Transcriptional Controls in the Regulation of Gene Expression, eds. Grunberg-Monago, M. & Safer, B. (Elsevier, New York), pp. 203-213. 33. Ayer, D. & Yarus, M. (1986) Science 231, 393-395. 34. Yamada, Y. & Ishikura, H. (1973) FEBS Lett. 29, 231-234. 35. Squires, C., Konrad, B., Kirschbaum, J. & Carbon, J. (1973) Proc. Natl. Acad. Sci. USA 70, 438-441. 36. Barrell, B. G. & Sanger, F. (1969) FEBS Lett. 3, 275-278. 37. Sprinzl, M. & Gauss, D. H. (1984) Nucleic Acids Res. 12, rl-r57. 38. Uhlenbeck, 0. C., Lowary, P. T. & Wittenberg, W. L. (1982) Nucleic Acids Res. 10, 3341-3352.
