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Restricted wobble in UGA codon recognition by glycine tRNA suppressors of UGG
J. Mol.
Biol.
(1981)
149, 1-13
Restricted Wobble in UGA Codon Recognition by Glycine tRNA Suppressors of UGG
Depurtment
of Molecular
M. D. Andrrson (Rrceil-rd
Biology,
Hospital 3 October
and
The University Tumor Institute,
of Texas Houston,,
1980, and irr revised form
System Cancer Center Tex. 77030, U.S.A.
19 January
1981)
In order to continue studies on the decoding properties in &vo of previously isolated UGG suppressor tRNAs, we devised selections for new mutations at two positions in trpA, the gene for t’he alpha subunit of tryptophan synthetase in Escherichia coli. The new mutants were used to test the response to them of several classes of UGG suppressors. In the presence of a gZ@derived suppressor of U(:A and UGG, we obtained the conversion of trpA(UAA211) to trpA(UGA211) and of trpA(UGU234) to both trpd(UGA234) and trpA(UGG234). The UGA mutants represent the first chainterminating mutants of that type to be verified in the tTp operon of E. co& The auxotrophic nature of trpA(UGG234) provides direct evidence that the presence of tryptophan at position 234 yields an inactive tryptophan synthetase alpha chain. Similarly, the failure of that mutant to be suppressed to prototrophy by U(X suppressors derived from supD supports the conclusion that an alpha chain with serine at position 234 is inactive. Finally, several of the UGG suppressors, derived from two different glycine tRNAs that read both GGA and GGG, fail to respond to UGA at one or both positions, even though they suppress both trpil(UGG211) and trp=l (UGG234). Those mutant tRNAs, therefore, in addition to having gained the new coding specificity have at least partially lost the ability to wobble in response to the third position of a codon in certain messenger RNA contexts. These results suggest the involvement of transfer RNA conformation or base modification in the specificit’y of codon recognition.
1. Introduction The wobble hypothesis proposed by Crick (1966) suggested a possible explanation not only of how fidelity is achieved in translation of the genetic code but also of one aspect of degeneracy, namely, how one transfer RNA might respond to more than one codon for the same amino acid. Despite conflicting results from studies irj vitro (e.g. see Mitra et al., 1977; Goldman et al., 1979: for a review, see Steege & S611, 1979). the basic predictions of the hypothesis are verifiable for non-mutant tRNAs in z+uo, at least for the glycine isoacceptors (Murgola & Pagel, 1980). Regarding mutationally altered tRNAs, many translational suppressors exist that represent codon specificit,ies and anticodon nucleotide sequence changes predictable from Crick’s hypothesis (for a review . see Smith, 1979). Such results, however, do not :i? 1981
Academic Press Inc. (London)
Ltcl.
2
E. .J. M/ll~KGOI,A
preclude the possibility that the tertiary structure of the entire tRNA molecule, and its interaction with the ribosome, might be important for the specificity of codon recognition (Kurland et al., 1975; Kurland, 1977,1979). Furthermore, several mutational studies have indicated the possible role of tRNA conformation in the determination of ribosome-dependent codon response (Hirsh, 1971; Buckingham $ Kurland, 1977; Vacher & Buckingham, 1979; Reeves & Roth, 1975; Pope et al.. 1978; Feinstein & Altman, 1977,1978; Murgola & Pagel, 1980). In previous work done by us and others, Escherichia coli missense suppressors were described that were derived from tRNA species that exhibit wobble in response.to the third position of the appropriate codons. In all cases in which the altered codon response involved the first, or second positions of the codon, the suppressors retained the third-position wobble. So, for example, GAA suppressors derived from glyV55, the gene for a GGA/G-reading mutationally altered form of gZyV tRNA (GGU/C-reader), respond also to GAG (Murgola & Yanofsky, 1974a; Murgola et al., 1978). Conversion of such GAA/G suppressors to suppressors of either UAA or UAG yielded tRNAs that respond to both UAA and UAG (Murgola & Jones, 1978; Murgola et al., 1978). The same has been true of mutants of glyT tRNA, the only glycine tRNA that responds to GGA (and GGG) in wild-type E. coli (Carbon et al., 1970; Murgola & Pagel, 1980). gZyT-derived suppressors of AGA, UGG and GAA respond also to AGG, LJGA, and GAG, respectively (Hadley & Murgola, 1978; Murgola & Childress, 1980; Murgola & Bryant, 1980). Furthermore, AAG suppressors derived from a gEyT(SuAGA/(:) respond to AAA in addition to ,4AG (E. J. Murgola, F. T. Page1 & K. H. Hadley, unpublished results). In contrast to those findings are results that we reported recently concerning the isolation in E. co& of several classes of UGG suppressors (Murgola & Childress, 1980). Of 11 gZyT-derived UGG suppressors, nine respond also to IJGA, as indicated by their ability to suppress a UGA mutant of phage T4. The other two, however, as well as all seven gZyV55-derived UGG suppressors, do not suppress that phage mutant. Although it could not be ruled out, misacylation did not seem likely as an explanation of these results since they involved nine independently occurring suppressors derived from two different tRNAs. Instead, we were intrigued by the possibility that at least some restriction of third-position wobble resulted from the generation of the suppressors, and even that such a wobble had been virtually entirely lost. Since both glyT and glyV55 tRNAs exhibit the third position A/G wobble, the biochemical study of such novel, essentially UGG-specific, suppressor tRNAs derived from each should provide interesting and important information concerning the involvement of tRNA conformation or base modification in codon recognition. We considered it necessary, however, to examine first the response of these suppressors to other UGA mutations, to bacterial in addition to phage mutations and, most important of all, to UGA codons occurring in the same position as the UGG codon. Consequently, this paper describes (1) the selection for a UGA codon that is “homotopic” (Feinstein & Altman, 1977) with the UGG codon previously used to select for the UGG suppressors, as well as for the same homotopic pair at another position in the same gene ; and (2) the use of these mutants to examine the response to UGA of several classes of UGG suppressors.
UGG-SPECIFIC
2. Materials
3
SUPPRESSORS
and Methods
(a) Bacterial strains In E. coli, trpA codes for the alpha subunit of tryptophan synthetase (TSase ; EC 4.2.1.20). A strain with a missense or nonsense mutation in the TSase alpha chain is designated trpA followed by parentheses containing the “mutant codon” and the number of the position of t,he amino acid substitution or chain-termination signal; for example, trpA(UGG211) or trpil(UAA211). In the strains used in this study, the trp genes, with specific trpA mutations, are contained in the cysB . h-p tonB region of the chromosome carried by the Fredericq (1969) episome, covering a chromosomal tonB-trp deletion. The construction of trpA mutant derivatives of the Fredericq episome has been described (Murgola et al., 1978). Reference to these derivatives will contain only the specific trpA mutation. Hence, F’trpA(UAA211) designates the entire Fredericq episome, with the normal alleles of all genes between cysB and tonB except for the trpA mutation in which the codon UAA occurs in place of GGA, corresponding to position 211 of the TSase alpha chain. The mutant trpA(UAA211) was obtained from trpA(GAA211) as described previously (Murgola & Jones, 1978), and trpA(UGU234) was originally designated trpA78 (Yanofsky et al., 1966). The isolation of the UGG or UGA/G suppressors used in this study has been described (Murgola & Childress, 1980). They were selected for their ability to suppress trpA(UGG211). Each is designated by a number preceded by EMS or SP, indicating that the suppressor either was induced with rthylmethanesulfonate or arose spontaneously. For use in this study, all suppressors were removed from the mutagenized or selective backgrounds. Other strains used in this study are all derivatives of E. coli K12 and aE described at first mention in Results. (b) Media
and bacteriological
procedures
With the use of the suppressor selection method (Berger & Yanofsky, 1967), spontaneously occurring UGA (and UGG) mutants were obtained as follows. Serial dilutions (in L-broth) of the appropriate strain (containing the trp region on the Fredericq episome) were incubated overnight at 37°C. In the morning, the highest dilution that grew w&8 washed in minimal salts, concentrated (3 or 4-fold), and plated on glucose minimal medium to select for Trp+ survivors. The colonies were screened to determine the nature of trpA aa follows. Prior to purification, each Trp+ colony was picked with a flat toothpick and transferred as a small patch to a fresh agar plate containing the same medium. Each patch plate, containing approx. 100 patches, was incubated at 37°C to allow growth of each prototroph. The fully grown patch plates were used as “masters” in replica-plate matings in order to transfer each episome to 2 different trp deletion strains, the first suppressor-free, the second containing a glyT-derived UGA/G suppressor. The desired trpA mutants (UGA or UGG) would result in a Trp- phenotype in the first mating but Trp+ in the second. Ind-BMT is glucose minimal medium containing a low concentration (15 pg/ml) of indole and a high concentration (50 pg/ml) of 5-methyl-m-tryptophan (5MT). The other media and genetic procedures used are described elsewhere (Murgola & Yanofsky, 19743) or in Results,
3. Results (a) Suppressor
selection
for
trpA(
UGil211)
Most of the well-characterized trpA mutations occur at position 211 of the TS alpha chain, where glycine (coded by GGA) is the wild-type amino acid. The mutations represent 23 codons including the two nonsense codons UAG and UAA (Yanofsky et al., 1969; Murgola & Yanofsky, 1974c; Murgola & Jones, 1978). The third nonsense codon, UGA, has not been obtained at that position or any other in trpd (nor has such a mutation been verified in any other gene of the trp operon). The absence of this class of nonsense mutation among trpA auxotrophs is somewhat
4
E. .J. MI’K(:C)lA
surprising since wild-type trpA has ten codons that could become UGA by a single base-change (Nichols & Yanofsky, 1979). Nevertheless. it seemed to us that, as long as UGA mutants of trp.4 are auxotrophic. we should be able to select for the conversion to UGA of existing trpA mutations (related to UGA by a single basechange) in the presence of a UGA suppressor. Such a suppressor, of course, would have to insert an amino acid that is functional at the mutant position of the protein. In the past, however, the well-characterized UGA suppressors were known either to insert tryptophan or to be derived from tryptophan tRNA (Chan & Garen, 1970: Hirsh, 1971 ; Ghan rt al., 1971 : Soll, 1974). Since there is no tryptophan residue in the wild-type TSase alpha chain, it is possible that the occurrence of that amino acid at any one of the ten positions rtprrsented by UGA-related codons results in a non-functional alpha chain (two positions have leucine. two serine, three cysteine, one arginine. and two glycine). If such were the case, it would not be possible to obtain or identify a U(:A muta,nt’ wit-h t,hose suppressors. Recently, however. UGA suppressors were obtained from a glycine tRNA (Murgola & Childress; 1980). In order to obtain trpA(~~GA211). we constructed the following strain: glyT(SuUGA/G) AtonB-trp/F’trpd (lTAA2I 1). it contains a glyT-derived suppressor of IT(:A and IJGG that was previously designated SP-11 (Murgola & Childress. 1980). The ochre mutation trp;l (FAA21 1) occurs on the Fredericq episome, which covers a chromosomal deletion from tonB to (at least) trpB. Since the enzymatic behavior is known for alpha chains containing each of 15 amino acids at position 211 (Murgola & Yanofsky. 1974c), one can predict precisely the nature of all prototrophs derivable from most trp.4 mutations at that position. Table 1 indicates the five possible single-step mutations in a frp-q(UAA211) strain that contains g/yT(SuUGA/G). In fact,, class B-2 was not considered likely to occur since the most frequent,ly occurring spontaneous ochre suppressors carry amino acids that. when inserted at position 211 of the alpha chain, yield a non-functional protein. Thus four classes remained. We were able to screen directly, however. for class B-3 as described in Materials and Methods. In that, fashion, several suppressed UGA were identified. After single-colony isolation, several mutants of trp.4 trp=l (UGA21 I)-containing episomes were transferred to a specifically marked. suppressor-free ton B-trp deletion strain.
Possihilitirs
for
single
step
prototrophic
trpAB/F’trpA A. trpA
dwivatiws of glyT(Su (I:=1 .d 21 I)
revertants
1. gZy?‘(SuUGA/G) 2. gZyT(SuUGA/G)
H. Suppressed
trpA
dtonR-trpAB/F’trpil(UCAIll) dtonB-trpAR/F’tr~(~~rU~ll)
mutants
I. glyT(SuUAA) dtonB-trpiiB/F’trpA(I.AA-“I 1) 2. SuU,4A gZyT(SuUGA/G) dtonB~tr~AB/F’trpA(ITAA~ll) 3. gZyT(SuUGA/G) dtonR-trpAB/F’tr~A(~GAZll)
UU=l/C?)
Aton-
UGG-SPECIFIC
(b) Suppressor
selection
for
6
SUPPRESSORS
trpA(
UGA234)
and
trpA(
UGG234)
Glycine is also the wild-type amino acid at position 234 of the alpha chain. One of the missense mutations affecting that position involves replacement of glycine by cysteine. It was formerly designated trpA78 and is due to the codon change of GGU to UGU (Yanofsky et al., 1966: Nichols & Yanofsky, 1979). We now refer to this mutant as trpA(UGU234). The existence of this mutant was particularly fortunate for us in that, in the presence of a UGA/G suppressor, it could theoretically undergo single-step mutations to both UGA and UGG. Until the present study, only at position 211 did a UGG mutation exist that was useful for selection of new suppressors (Murgola & Yanofsky, 1974c; Murgola & Childress, 1980). Indirect evidence, however, suggested that tryptophan is not functional at position 234. The parent strain constructed in order to obtain the new mutations at position 234 was gZyT(SuUGA/G) dtonBtrpAB/F’trpA(UGU234). Since not as much is known about amino acid replacements at positon 234 as at position 211: we cannot make a list of all possible single-step prototrophic derivatives of the parent strain, as we did in the previous section for position 211 (Table 1). But when codon-specific suppressors exist, that lack of information is no handicap. Consequently, we screened prototrophic derivatives of trpA(UGU234) as described in Materials and Methods, with one exception. In the replica-plate matings to transfer out the episomes. between the first mating (into a suppressorfree st’rain) and the final mating (into the UGA/G-suppressor strain) we included a mating into a strain containing a gZyEderived UGG suppressor (Murgola $ (‘hildress, 1980). glyU codes for a glycine tRNA that responds to GGG but not (EA. The rGG suppressor derived from it does not suppress a phage T4 UGA mutation (one that is suppressed by other glycine tRNA UGG suppressors), and so appears to be UGG-specific, as expected. In the screening, F’trpA(UGG234) was identified by yielding Trp+ exconjugants in both the second and third matings. whereas F’trpA((UGA234) did so only in the third mating. After single-colony isolation, isolates of each type were verified by individual matings.
(c) Growth
of trpA mutants, suppressed on indolef5-methyl-nL-tryptophun
and unsuppressed, medium
Missense mutants of trpA make a complete, although enzymatically inactive, TSase alpha chain. Such mutants produce an alpha chain that can activate the TSase beta subunit in the conversion of indole to tryptophan. They are able, consequently, to grow on glucose minimal medium containing a low concentration (15 pg/ml) of indole and a high concentration (50 pg/ml) of 5-methyl-nLtryptophan (Ind-5MT). In the presence of 5MT, which acts as a corepressor, the trp operon is fully repressed. In this condition, a low amount of the beta chain is made, but it is insufficient to allow the bacterium to grow on 1.5 pg of indole per ml unless the bacterium also produces a complete alpha chain that can activate whatever amount of beta chain is made. Consequently, since nonsense trpA mutants do not make a complete alpha chain that can activate the beta chain, they cannot grow on Ind-5MT.
6
E. I. MURGOLA
Such reasoning could lead one to expect that, in the presence of a nonsense suppressor of the appropriate codon specificity, a given trpA nonsense mutant would grow on Ind-5MT. We found this to be the case (Murgola & Jones, 1978) in the suppression of trpA(UAG211) by the amber suppressors supD and ,supF, which insert serine and tyrosine, respectively. However, when we tested trpA(UAA211) and trpA(UAG211) in the presence of an ochre suppressor derivative of supD, the strains did not grow on Ind-5MT (Murgola & Jones, 1978). We considered this result reasonable because ochre suppressors are generally much less efficient than amber suppressors. We suggested that any trpA nonsense mutant in the presence of a corresponding low-efficiency suppressor does not produce enough complete alpha chains in the repressed state of the trp operon (i.e. in the presence of 5MT) to activate the beta chains sufficiently to allow growth on Ind-5MT. The experimental basis for the discussion in the previous two paragraphs involved only amber and ochre mutants because until the present study no UGA mutation in trpA had been identified. Consequently, we were interested in determining whether the two trpA UGA mutants can grow on Ind-SMT, in the presence or absence of the UGA suppressor SP-11 and others recently isolated by US. As determined either by replica-plating of patches or by streak outs from cell suspensions, neither trpA(UGA211) nor trpA(UGA234) was able to grow on Ind5MT. As expected, however, trpA(UGG234), being a missense mutant, was able to grow on that medium. Growth of the UGA mutants in the presence of various UGA suppressors is reported in the next section. (d) Suppressor
response
to UCA
and UGG at two positions
in trpA
Recently (Murgola & Childress, 1980), we isolated several classes of UGG suppressors using the mutant trpA(UGG211). Some did not suppress a certain UGA mutant of phage T4 and so appeared to represent UGG-specific suppressors, that is, ones that do not exhibit “wobble” with regard to the third position of the codon. Especially interesting were those derived from tRNAs that do exhibit such wobble, namely gZyl’55 and gZyT (see Table 2). To test this loss of wobble using bacterial TABLE tRlVA Gene symbol
genes from
which supn
UCX
2
suppressors
were obtained
C&V55
mutationally
SYT
SlYU
Codon recognized (5’ 43’1
UAG
Anticodon (3’+5’)
-a
GGA.
GGG
CCl’b
a supD tRNA is derived from a minor swine tRNA ant.irodon (D. Steege, personal communication). b Squires & Carbon (1971). ’ Roberts & Carbon (1975); firepresents an unidentified the gZyT tRNA molecules isolated from B. roli veils. d Hill ef al. (1973).
GGA,
GGG
GGG
C&F
by a single
derivative
ccc*
base change
of U that
in the middle
is present
of the
in a portion
of
glyV5P
SP-I. SP-3, SP-5, SP-6,
+
+ +
+
-
-
+
-
+
+
+e
Mind
ITGG2 11
-
Suppression of a phage T4 C’GA mutantC
-
+
-
+
-
-
-e
Min
EGA21
-
-
-
+
-
Tnd-5MTd
1
+
+
+
+
+
-
Min
UGG”34
-
-
-
a For origin and nomenclature, see Materials and Methods. bSee Table 2 for codon specificities and anticodons. ’ From Murgola & Childress (1980). dMin, glucose minimal medium; Ind-5MT, Min plus indole(l.5 pg/ml) and 5.methyl-DI.-tryptophan (50 &ml). e + , growth, and -, no growth, on Min or Ind-BMT after 5 days of incubation at 37°C. ‘This suppressor arose in a supD-containing strain, is linked to his, retains supD activity. and so far has not been separated B A mutant form of gZyV, which codes for a GGU/C-reading glycine tKNA.
glyV55
c&d’
SP-13
SP-9,
SP-14
sW”
EMS-B, EMS-e, EMS-7, EMS-& EMS-I), SP-11, SP-18, SP-19, SP-20
SP-4, SP-7
sly CT
SP-15
supD
?
EMS-2 EMS-4’
Parental tRNA geneb
SP-12’
EMS-l EMS-31
Suppressors examined”
from
supD.
-
-
genetically
-
-
-
In&5MT
-
+
-
Min
UGA234
8
E. ,I. MUR(:OLA
mutations, we obtained two homotopic pairs of UGA and UGG mutations, at TSase alpha chain positions 211 and 234, as described in the first two sections of Results. We then constructed tonB-trp deletion strains containing representatives of each of several categories of those suppressors. We emphasize that each suppressor was first removed, by transduction, from its original mutagenized or selective genetic background. Through conjugation, Fredericq episomes carrying the four trpA mutations (UGA and UGG, at positions 211 and 234) were introduced into each suppressor-containing trp deletion strain by selecting for growth on glucose minimal medium plus indole (trpB). Finally. suppression was determined by testing the exconjugants for growth on glucose minimal medium (Min) and on Min plus low (1.5 pg/ml) indole and high (50 pg/ml) 5-methyl-uL-tryptophan (Ind-5MT). The results are shown in Table 3. Neither SP-12 nor any of the suppressors derived from supD or glyU exhibit suppression of either UGA mutation. Also, whereas SP-15 suppresses trpL4(UGG234), SP-12 and EMS-l, EMS-Z, EMS-3, and EMS-4 do not. trpA(UGA211) is suppressed by six gZyV55-derived suppressors and nine glyT-derived ones. Those nine gEyT suppressors, however, are unique in two respects: (1) only they suppress trpA(UGA234) on Min; and (2) only they allow trpA(UGA211) to grow on Ind-5MT, although they do not do so for trpA(UGA234). All st.rains containing trpA(UGG211) or trpA(UGG234) grow on Ind$MT, even in t*he absence of any suppressor (not shown in Table 3). After introduction of each episome into each suppressor-containing trp deletion strain, all of the resulting strains were not only tested for growth on Min and Ind5MT but also examined as follows. To show the presence of the proper trpA mutation, the episome was transferred by mating into appropriate suppressor strains. To verify the functional presence of the correct suppressor two things were done. (1) For those strains that did not’ grow on Min, F’trp.4 (UGG211) was mated in and the exconjugants tested for growth on Min. (2) Phage Pl lysates were made on all strains and then tested for the correct suppressor by transductional linkage to the proper marker (purrl, metB. @A and his).
4. Discussion In this paper we have reported (1) the isolation of trpA mutants representing UGA at TSase alpha chain positions 211 and 234, as well as UGG at position 234; and (2) the use of these new mutants to test the response to them of several classes of UGG suppressors. Until now, no UGA nonsense mutations have been found in trpA or verified in any other trp operon gene in E. coli. The recent isolation, however, of a UGA suppressor derived from a glycine tRNA (Murgola & Childress, 1980) provided the opportunity to look for UGA at alpha chain positions where glycine is the wild-type amino acid. At position 211 the glycine codon is GGA, one of the ten trpA codons related to UGA by a single base change. The glycine codon at 234 is GGU and would not have yielded a single-step UGA mutation. Using the suppressor selection method, we readily obtained UGA mutations at both positions.
UGG-SPECIFIC
SUPPRESSORS
9
Despite previous observations concerning UGA mutations in E. coli and Salmon& (e.g. see Roth, 1970), the new mutants trpA(UGA211) and trpA (UGA234) are not leaky ; they are cleanly TrpThis is true in strains that are not streptomycin resistant and have no known ribosomal mutations (that is. mutations that might result in restriction of some hypothetical low-level UGAsuppressing activity). These mutants also do not grow on Ind-5MT medium (see Materials and Methods) as is true of all nonsense mutants (UAA and UAG) of trpA examined thus far (Murgola & Yanofsky, 1974c; Murgola & Jones, 1978). The failure of trpA(UGA211) to grow on that medium in the presence of six gZyVn’5derived UGA suppressors indicates that the efficiency of those suppressors is very low compared with the nine g&T UGA suppressors and with the highly efficient amber suppressor supD (Murgola & Jones, 1978). In Results section (a), above, we discussed the possibility that previous studies may not have detected UGA mutations due to the amino acid specificity of the existing suppressors. Among the trp operon mutants obtained by Yanofsky and coworkers, there are some that were determined to be due to chain-terminating mutations but were not suppressed by several different nonsense suppressors (see. e.g. Yanofsky et al., 1971). But generally only one UGA suppressor was employed, a tryptophan-inserting one. Therefore, some of those unidentified-chain-terminators may be UGA mutants. Consequently, testing them with one or several of our new ITGA suppressors might identify a variety of useful and perhaps very interesting ITUA mutations. All three nonsense codons now exist at position 2 11, and the presence of UGA at position 234 allows us to select for UAA and UAG in t,he presence of ochre and amber-specific suppressors, respectively. Furthermore, since the UGA selection worked in these two instances it should be possible to obtain UGA, as well as UAA and UAG, at other positions in trpA , regardless of whether they are presently sites of nonsense or missense mutations. Having this homotopic triad of nonsense codons at, many positions within the same well-characterized gene will make possible the desirable extension of studies begun (Feinstein & Altman, 1977,1978; Bossi & Roth, 1980: Fluck & Epstein, 1980) on the effects of mRNA context and tRNA structure on the process of translation of the genetic code. Some years ago, an indication of the non-functionality of alpha chains carrying tryptophan or serine at position 234 was provided by indirect evidence, namely the absence of tryptophan or serine-substituted prototrophic revertants of trpA(UGU234), formerly designated trpA78 (Yanofsky et al., 1966). In the present work, direct evidence for the behavior of tryptophan was obtained by our ability to generate trpA (UGG234) as an auxotroph. Furthermore (data not presented), trp=l (UGA234) is not suppressed to Trp+ by a tryptophan-inserting UGA suppressor (Sambrook et al., 1967). The same is true of trpA4(UGA21 l), but that was expected on the basis of the known amino acid requirements of alpha chain position 211 (Murgola B Yanofsky, 1974c). Less conclusive, however, is the result concerning serine, namely that suppression of trpA(UGG234) to prototrophy was not exhibited by the four UGG suppressors derived from the serine-inserting amber suppressor supD. The force of the conclusion is weakened by the fact, that we cannot’ be certain that the UGG-reading forms of that tRNA still insert serine.
10
E. .J. MCRGOLA
Furthermore, the failure to suppress trpA(UGG234) as opposed to trpil(UGG211) could be due to the difference of the nucleotide sequence “context” of their mRNA codons (Nichols & Yanofsky, 1979) rather than to the nature of the amino acid inserted. Once trpA (UAG234) is obtained, however, it should be possible to provide definitive evidence by demonstrating that supD itself, known to insert serine, relieves chain termination at that trprl nonsense codon and produces a complete but’ enzymatically inactive alpha chain (Murgola & Yanofsky, 1974c; Murgola $ Jones, 1978). The inability of the supD-derived UGG suppressors to respond to UGA is not surprising since supD tRNA reads UAG but not UAA (see Table 2). A similar comment can be made about gly U suppressor SP-15, which is derived from a glycine tRNA that reads GGG but not GGA (Carbon et al., 1970; Murgola & Pagel, 1980). Since the identity of the his-linked suppressor SP-12 is unknown, no prediction was possible regarding its response to UGA. As it turns out, it does not suppress either UGA mutation or trpA(UGG234). SP-12 could be derived from a cysteine tRNA gene that is located in the supD region of the chromosome (Ikemura & Ozeki, 1977). It is known that the occurrence of cysteine at position 234 yields a non-functional alpha chain (Yanofsky et al., 1966). Finally, another category that was understandably anticipated comprises SP-11 and eight other glyT suppressors that respond to all three UGA mutations. Since they are derived from a species that reads A or G in the third position of the codon, we expected that such UGG suppressors would also suppress UGA. Surprising results were obtained, however, with the remaining suppressors (two derived from glyT, and seven from glyV%), and three considerations arising from their behavior implicate aspects of tRNA structure other than the anticodon in the specificity of codon recognition. First, since glyT and glyV55 each gave rise to at least two classes of UGG suppressors, at least two kinds of single nucleotide change must be involved in each case. Therefore, at least one type of change must be one that is not predictable according to classical codon-anticodon hydrogen-bonding rules. Second, although the tRNAs coded by glyT and glyV55 respond to GGA in addition to GGG, two suppressors derived from the former and one from the latter fail to suppress the two bacterial UGA mutations in addition to the phage T4 mutation. The fact that all three suppress homotopic UGG codons at two positions in trpA (211 and 234) shows that misacylation is not involved in the failure to suppress the bacterial UGA mutations. It must be kept in mind that the conversion of any glycine tRNA to a UGG suppressor involves primarily a change in response to the first position of the codon. But’ in the case of the UGA-restrictive suppressors. any such change, whether it is in the anticodon or elsewhere in the molecule, must also effect the third-position response. Consequently, an effect on tRNA conformation is indicated. We consider it unlikely that these suppressors are due to two nucleotide changes having occurred in one mutational event. First, the frequency of their occurrence is high (Murgola & Childress, 1980; and unpublished data). Second, only one type of single-step translational suppressor has been shown to be due to two nucleotide changes, and that kind occurs at a very low frequency and in response to nitrosoguanidine or ultraviolet irradiation (Coleman et al., 1980). We cannot rule out. however, the possible involvement of a cryptic mutation in a
UGG-SPECIFIC
SUPPRESSORS
11
T-ABLE 4 Messenger
RIvA
Alpha
position
chain
mKNA
codons
Amino
acids
t Nichols
codon contexts? the tryptophun 209 UUG Leu
& Yanofsky
of UGA mutations corresponding to two position,s synthetase alpha chain of E. coli 210
211
CAG
I’GA
Gln
.- -
212 UUC Phe
313.. GGU Gly
232 9tW Ile
233 UCI’ Ser
234
235
LTGA UCG --~
Ser
in
236.. GCC Ala...
(1979)
closely linked gene that codes for a tRNA-related molecule (for example, a tRNAmodification enzyme). The third consideration concerning tRNA structure and codon recognition is mRNA context effects. Six of the suppressors derived from gZyV55 suppress trpA(UGA211) but not trpA(UGA234), although they all suppress UGG at both positions. Also, in the presence of the nine gZyT UGA suppressors, trpA(UGA211) is suppressed sufficiently to grow on Ind-5MT, but trpA(UGA234) is not. The nucleotide sequences in the neighborhood of UGA211 and UGA234 are clearly different (Nichols & Yanofsky, 1979; and Table 4). Nevertheless, neither region displays features that bear any obvious relation to previous findings concerning suppression of UAA or UAG in different contexts (Feinstein & Altman: 1977,197s: Bossi & Roth, 1980). Such differential context-dependent responses to a given codon by one and the same tRNA, observed now for all three nonsense codons. cannot be due simply to codon-anticodon hydrogen bonding. The influence of the. nucleotides adjacent to the codon could be by way of an effect on mRNA secondary structure (Grosjean et aE., 1978; Grosjean & Chantrenne, 1980). Alternatively, it may involve differential interactions of the suppressor tRNA with those nucleotides or with the ribosome, an adjacent tRNA, or release factors. Finally, the effect might be manifested after peptide bond formation in the stability of the peptidyl-tRNA (Caplan & Menninger, 1979), or in the generation of shifts in reading frame (Atkins et al., 1979; Kurland, 1979). We have not yet determined, by enzyme assays, the efficiencies of suppression of trpA(UGG211) and trpA(UGG234) by any of the suppressors. Nevertheless, those that display context-dependent ITGA suppression show no gross difference in suppression of UGG at the two positions as judged by rates of growth in liquid medium and colony size on solid medium (data not presented). This fact suggests that in these cases, the context effects relate more to factors involved in chain termination than to parameters such as tRh’A-tRNA interaction. It will be interesting to investigate whether any missense suppressors display context effects in their response to sense codons. The isolation of glyT- and gZyV55-suppressor tRNAs for nucleotide sequence analysis is underway. In the primary-structure analysis we are concerned first with the location and nature of the mutationally determined nucleotide change. Of special interest is the anticipation that some suppressors might be due to mutations quite distant from the anticodon, as in the case of the well-studied tryptophan
12
E. ,I. MUKGOLA
tRNA UGA suppressor (Hirsh, 1971). But we must also ask whether any change in base modification has occurred. Specifically. is the adenine that is adjacent to the anticodon on the 3’ side modified with an isopentenyl moiety? If so. to what extent? Is the modified uracil in the 5’ position of the anticodon of gEyT tRNA (see Table 2) under-modified or modified differently? Has the uracil in the 5’ position of the anticodon of gZyV55 tRNA, or any other normally unmodified base in either species, become modified as a result of the primary structure change? Particularly intriguing is the question of the nature of the changes that restrict third position wobble (this paper) or allow.gZyT UGA suppressors to exhibit an unorthodox wobble with regard to the first position of a codon (Murgola & Pagel, 1980). Once we have t,he answers to these questions. we may be able to make specific correlations between tRNA structure and the specificity of codon recognition. In any event, it is clear in general, from the present study and previous work of others, that parts of the tRNA structure other than the primary anticodon sequence must be involved in the process of codon recognition, whether it be by way of nucleotide modifications or conformational changes of the tRNA molecule (see Introduction, and also Mijller et al., 1979). In the light of those studies and recent, ones on mitochondrial tRNAs (Barrel1 et al., 1979,198O ; Bonitz et aZ., 1980 : Eperon et a,l., 1980; Heckman rt al.. 1980). it has become necessary to rethink the “rules” for codon recognition by transfer RNAs. One may wonder, however, how likely it is that any universal rules can be formulated that will apply uniformly to all tRNAs. For excellent technical assistance with different portions of this work, 1 am most grateful t,o Frances Pagel, Kathryn Hijazi and Patrick Condreay. I also thank Charles Yanofsky for his comments on the manuscript and Walter Pagel, Janet Naquin and Janie Finch for assistance in its preparation. Portions of this work were supported by grants from the American Cancer Society (NP-167) and the U.S. Public Health Service (GM 21499, from the National Institute of General Medical Sciences). The author is the recipient of U.S. Public Health Service research career development award GM 00047 from The National Institute of General Medical Sciences.
REFERENCES Atkins, J. F., Gesteland, R. F., Reid, B. R. & Anderson, C. W. (1979). CeZl, 18, 1119-1131. Barrell, B. G., Bankier, A. T. & Drouin, J. (1979). Nature (London), 282, 189-194. Barrell, B. G., Anderson, S., Bankier, A. T., de Bruijn, M. H. L., Chen, E., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R. & Young, I. G. (1980). Proc. ,Vat. Acud. Sci., U.S.A. 77,3164-3166. Berger, H. & Yanofsky, C. (1967). Science, 156, 394-397. Bonitz, S. G., Berlani, R., Coruzzi, G., Li, M., Macino, G., Nobrega, F. G., Nobrega, M. P., Thalenfeld, B. E. & Tzagoloff, A. (1980). Proc. Nat. Acad. Sci., U.S.A. 7’7, 3167-3170. Bossi, L. & Roth, J. R. (1980). A’ature (London), 286, 123-127. Buckingham, R. H. & Kurland, C. G. (1977). Proc. IVat. Acud. Sci., U.S.A. 74, 5496-5498. Caplan, A. B. & Menninger, J. R. (1979). J. Mol. Biol. 134, 621-637. Carbon, J., Squires, C. & Hill, C. W. (1970). J. Mol. Biol. 52, 571-584. Chan, T. & Garen, A. (1970). J. Mol. Biol. 49, 231-234. Chan, T., Webster, R. E. & Zinder, N. D. (1971). J. Mol. BioE. 56, 101-116.
CGG-SPECIFIC
SI:PPKESSOR~
I3
Coleman. R. D., Dunst, R. W. & Hill, C. W. (1980). Mol. (:en. Drnet. 177, 213-222. Crick, F. H. C. (1966). J. Mol. Biol. 19, 548-555. Eperon. 1. C.. Anderson, S. & Nierlich, D. P. (1980). Suture (London). 286, 46&467. Feinstein. S. I. &, Altman, S. (1977). J. Mot. Biol. 112, 453-470. Feinstein, S. I. & Altman, S. (1978). Urn&s, 88, 201-219. Fluck, M M. & Epstein, R. H. (1980). Mol. Gen. Genet 177. 615627. Fredericq, P. (1969). In Bacterial Plasmids and Episomrs (Wolstenhome. (:. E. W. 8r O’Connor, M., eds), pp. 1633174, Little, Brown and Co.. Boston. (Goldman. E.. Holmes, W. M. & Hatfield. G. W. (1979). J. Mol. Biol. 129, 567-585. (irosjean. H. $ Chantrenne, H. (1980). In Chemical Recognitiorc in Biology (Chapeville, F. 8i Haenni, A.-L.. eds), pp. 3477367, Springer-Verlag, Berlin. (irosjean. H. J.. de Henau. S. & Crothers, D. M. (1978). Proc. Sat. =Icad. Sci.. 1I.S.d. 75. 610-614. Hadley, K. H. & Murgola, E. J. (1978). (‘urr. ~~icrohiol. 1, 99-103. Heckman. ,J. E., Sarnoff, J., Alzner-DeWeerd, Tin. S. & RajBhandary. U. L. (1980). Proc. .\rut. .jcad. Sk., U.S.A. 77: 315993163. Hill. C. WT., (‘ombriat.o, G.. Steinhart. W.. Riddle. D. L. & Carbon, J. (1973). J. Biol. Chrm. 248. 42.X-4262, Hirsh, D. (1971). J. Mol. Biol. 58, 439-458. Ikemura, T. & Ozeki, H. (1977). J. Mol. Biol. 117, 419-446. Kurland, C. G. (1977). dnnu. Rev. Biochem. 46, 173-200. Kurland, C. G. (1979). In AVonsense Mutations and tR,VA Suppressors (Celis, J. E. & Smith. ,J. D.. eds). pp. 977108, Academic Press, New York and London. Kurland, C. C:., Rigler, R.. Ehrenberg, M. & Blomberg, C. (1975). Proc. ivat. Acud. Sei.. l’.S..1. 72, 424884251. Mitra. S. K., Lustig, F., Akesson, B.. Lagerkvist, U. & Strid. L. (1977). J. Biol. Chem. 252, 471-478. Miiller. A., Mandershied, U., Lipecky, R., Bertram, S., Schmitt, M. & Gassen, H. G. (1979). In 7kxnsfer RSA: Structure, Properties, and Recognition (Schimmel, P. R., Still, D. & Abelson, J. N., eds), pp. 4599471, Cold Spring Harbor Laboratory, New York. Murgola. E. ,J. & Bryant, J. E. (19801. J. Bucteriol. 142, 131-137. Murgola. E. ,J. & Childress, J. R. (1980). J. Bacterial. 143, 285-292. Murgola. E. ,J. & Jones, C. I. (1978). Mol. Gen. Genet. 159, 179184. Murgola. E. ,J. &, Pagel, F. T. (1980). J. Mol. Biol. 138, 833-844. Murgola. E. ,J. & Yanofsky, C. (1974a). J. Bacterial. 117, 439-443. Murgola. E. J. & Yanofsky, C. (19746). J. Bacterial. 117, 444-448. Murgola. E. ,J. 8: Tanofsky, C. (1974c). J. Mol. BioZ. 86, 775-784. Murgofa. E. ,I., Prather, N. E. & Hadley, K. H. (1978). J. Bacterial. 134, 801-807. Nichols? B. P. & Yanofsky, C. (1979). Proc. Sat. ilcad. Sci., I!.S.=l. 76, 52445248. Pope. W. T., Brown. A. & Reeves. R. H. (1978). Xucl. Acids Res. 5. 104-1057. Reeves. R. H. & Roth, J. R. (1975). J. Bacterial. 124, 332-340. Roberts. ,J. W. & Carbon, J. (1975). J. Biol. Chem. 250, 553&5541, Roth. J. K. (1970). J. Bacterial. 102. 467-475. Sambrook, *I. F., Fan, D. P. & Brenner, S. (1967). ,Vature (London), 214, 452-453. Smith. ,J. D. (1979). In Sorrsense Mutations and tRIVA Suppressors (Celis, J. E. & Smit.h. ,J. D.. eds), pp. 109125, $cademic Press, New York and London. Sol], L. (1974). J. Mol. Biol. 86, 2333243. Squires. C. & Carbon, J. (1971). Nature Sew Biol. 233, 274-277. Steege. D. A. & Still, D. G. (1979). In Biological Regulation and Development (Goldberger, R. F.. ed.), vol. 1, pp. 4333485, Plenum Publishing Corp., New York, Vaeher. J. & Buckingham. R. H. (1979). J. Mol. Biol. 129, 287-294. Yanofsky. C.. Ito, J. & Horn, V. (1966). CoZd Spring Harbor Symp. f&ant. Biot. 31, 151-162. Yanofsky, C., Berger, H. & Brammar, W. J. (1969). Proc. XII Int. Coqr. Genet. 3, 155-165. Tanofsky. (‘.. Horn. V., Bonner, M. & Stasiowski. 6. (1971). C:enetics, 69, 409433.
Biol.
(1981)
149, 1-13
Restricted Wobble in UGA Codon Recognition by Glycine tRNA Suppressors of UGG
Depurtment
of Molecular
M. D. Andrrson (Rrceil-rd
Biology,
Hospital 3 October
and
The University Tumor Institute,
of Texas Houston,,
1980, and irr revised form
System Cancer Center Tex. 77030, U.S.A.
19 January
1981)
In order to continue studies on the decoding properties in &vo of previously isolated UGG suppressor tRNAs, we devised selections for new mutations at two positions in trpA, the gene for t’he alpha subunit of tryptophan synthetase in Escherichia coli. The new mutants were used to test the response to them of several classes of UGG suppressors. In the presence of a gZ@derived suppressor of U(:A and UGG, we obtained the conversion of trpA(UAA211) to trpA(UGA211) and of trpA(UGU234) to both trpd(UGA234) and trpA(UGG234). The UGA mutants represent the first chainterminating mutants of that type to be verified in the tTp operon of E. co& The auxotrophic nature of trpA(UGG234) provides direct evidence that the presence of tryptophan at position 234 yields an inactive tryptophan synthetase alpha chain. Similarly, the failure of that mutant to be suppressed to prototrophy by U(X suppressors derived from supD supports the conclusion that an alpha chain with serine at position 234 is inactive. Finally, several of the UGG suppressors, derived from two different glycine tRNAs that read both GGA and GGG, fail to respond to UGA at one or both positions, even though they suppress both trpil(UGG211) and trp=l (UGG234). Those mutant tRNAs, therefore, in addition to having gained the new coding specificity have at least partially lost the ability to wobble in response to the third position of a codon in certain messenger RNA contexts. These results suggest the involvement of transfer RNA conformation or base modification in the specificit’y of codon recognition.
1. Introduction The wobble hypothesis proposed by Crick (1966) suggested a possible explanation not only of how fidelity is achieved in translation of the genetic code but also of one aspect of degeneracy, namely, how one transfer RNA might respond to more than one codon for the same amino acid. Despite conflicting results from studies irj vitro (e.g. see Mitra et al., 1977; Goldman et al., 1979: for a review, see Steege & S611, 1979). the basic predictions of the hypothesis are verifiable for non-mutant tRNAs in z+uo, at least for the glycine isoacceptors (Murgola & Pagel, 1980). Regarding mutationally altered tRNAs, many translational suppressors exist that represent codon specificit,ies and anticodon nucleotide sequence changes predictable from Crick’s hypothesis (for a review . see Smith, 1979). Such results, however, do not :i? 1981
Academic Press Inc. (London)
Ltcl.
2
E. .J. M/ll~KGOI,A
preclude the possibility that the tertiary structure of the entire tRNA molecule, and its interaction with the ribosome, might be important for the specificity of codon recognition (Kurland et al., 1975; Kurland, 1977,1979). Furthermore, several mutational studies have indicated the possible role of tRNA conformation in the determination of ribosome-dependent codon response (Hirsh, 1971; Buckingham $ Kurland, 1977; Vacher & Buckingham, 1979; Reeves & Roth, 1975; Pope et al.. 1978; Feinstein & Altman, 1977,1978; Murgola & Pagel, 1980). In previous work done by us and others, Escherichia coli missense suppressors were described that were derived from tRNA species that exhibit wobble in response.to the third position of the appropriate codons. In all cases in which the altered codon response involved the first, or second positions of the codon, the suppressors retained the third-position wobble. So, for example, GAA suppressors derived from glyV55, the gene for a GGA/G-reading mutationally altered form of gZyV tRNA (GGU/C-reader), respond also to GAG (Murgola & Yanofsky, 1974a; Murgola et al., 1978). Conversion of such GAA/G suppressors to suppressors of either UAA or UAG yielded tRNAs that respond to both UAA and UAG (Murgola & Jones, 1978; Murgola et al., 1978). The same has been true of mutants of glyT tRNA, the only glycine tRNA that responds to GGA (and GGG) in wild-type E. coli (Carbon et al., 1970; Murgola & Pagel, 1980). gZyT-derived suppressors of AGA, UGG and GAA respond also to AGG, LJGA, and GAG, respectively (Hadley & Murgola, 1978; Murgola & Childress, 1980; Murgola & Bryant, 1980). Furthermore, AAG suppressors derived from a gEyT(SuAGA/(:) respond to AAA in addition to ,4AG (E. J. Murgola, F. T. Page1 & K. H. Hadley, unpublished results). In contrast to those findings are results that we reported recently concerning the isolation in E. co& of several classes of UGG suppressors (Murgola & Childress, 1980). Of 11 gZyT-derived UGG suppressors, nine respond also to IJGA, as indicated by their ability to suppress a UGA mutant of phage T4. The other two, however, as well as all seven gZyV55-derived UGG suppressors, do not suppress that phage mutant. Although it could not be ruled out, misacylation did not seem likely as an explanation of these results since they involved nine independently occurring suppressors derived from two different tRNAs. Instead, we were intrigued by the possibility that at least some restriction of third-position wobble resulted from the generation of the suppressors, and even that such a wobble had been virtually entirely lost. Since both glyT and glyV55 tRNAs exhibit the third position A/G wobble, the biochemical study of such novel, essentially UGG-specific, suppressor tRNAs derived from each should provide interesting and important information concerning the involvement of tRNA conformation or base modification in codon recognition. We considered it necessary, however, to examine first the response of these suppressors to other UGA mutations, to bacterial in addition to phage mutations and, most important of all, to UGA codons occurring in the same position as the UGG codon. Consequently, this paper describes (1) the selection for a UGA codon that is “homotopic” (Feinstein & Altman, 1977) with the UGG codon previously used to select for the UGG suppressors, as well as for the same homotopic pair at another position in the same gene ; and (2) the use of these mutants to examine the response to UGA of several classes of UGG suppressors.
UGG-SPECIFIC
2. Materials
3
SUPPRESSORS
and Methods
(a) Bacterial strains In E. coli, trpA codes for the alpha subunit of tryptophan synthetase (TSase ; EC 4.2.1.20). A strain with a missense or nonsense mutation in the TSase alpha chain is designated trpA followed by parentheses containing the “mutant codon” and the number of the position of t,he amino acid substitution or chain-termination signal; for example, trpA(UGG211) or trpil(UAA211). In the strains used in this study, the trp genes, with specific trpA mutations, are contained in the cysB . h-p tonB region of the chromosome carried by the Fredericq (1969) episome, covering a chromosomal tonB-trp deletion. The construction of trpA mutant derivatives of the Fredericq episome has been described (Murgola et al., 1978). Reference to these derivatives will contain only the specific trpA mutation. Hence, F’trpA(UAA211) designates the entire Fredericq episome, with the normal alleles of all genes between cysB and tonB except for the trpA mutation in which the codon UAA occurs in place of GGA, corresponding to position 211 of the TSase alpha chain. The mutant trpA(UAA211) was obtained from trpA(GAA211) as described previously (Murgola & Jones, 1978), and trpA(UGU234) was originally designated trpA78 (Yanofsky et al., 1966). The isolation of the UGG or UGA/G suppressors used in this study has been described (Murgola & Childress, 1980). They were selected for their ability to suppress trpA(UGG211). Each is designated by a number preceded by EMS or SP, indicating that the suppressor either was induced with rthylmethanesulfonate or arose spontaneously. For use in this study, all suppressors were removed from the mutagenized or selective backgrounds. Other strains used in this study are all derivatives of E. coli K12 and aE described at first mention in Results. (b) Media
and bacteriological
procedures
With the use of the suppressor selection method (Berger & Yanofsky, 1967), spontaneously occurring UGA (and UGG) mutants were obtained as follows. Serial dilutions (in L-broth) of the appropriate strain (containing the trp region on the Fredericq episome) were incubated overnight at 37°C. In the morning, the highest dilution that grew w&8 washed in minimal salts, concentrated (3 or 4-fold), and plated on glucose minimal medium to select for Trp+ survivors. The colonies were screened to determine the nature of trpA aa follows. Prior to purification, each Trp+ colony was picked with a flat toothpick and transferred as a small patch to a fresh agar plate containing the same medium. Each patch plate, containing approx. 100 patches, was incubated at 37°C to allow growth of each prototroph. The fully grown patch plates were used as “masters” in replica-plate matings in order to transfer each episome to 2 different trp deletion strains, the first suppressor-free, the second containing a glyT-derived UGA/G suppressor. The desired trpA mutants (UGA or UGG) would result in a Trp- phenotype in the first mating but Trp+ in the second. Ind-BMT is glucose minimal medium containing a low concentration (15 pg/ml) of indole and a high concentration (50 pg/ml) of 5-methyl-m-tryptophan (5MT). The other media and genetic procedures used are described elsewhere (Murgola & Yanofsky, 19743) or in Results,
3. Results (a) Suppressor
selection
for
trpA(
UGil211)
Most of the well-characterized trpA mutations occur at position 211 of the TS alpha chain, where glycine (coded by GGA) is the wild-type amino acid. The mutations represent 23 codons including the two nonsense codons UAG and UAA (Yanofsky et al., 1969; Murgola & Yanofsky, 1974c; Murgola & Jones, 1978). The third nonsense codon, UGA, has not been obtained at that position or any other in trpd (nor has such a mutation been verified in any other gene of the trp operon). The absence of this class of nonsense mutation among trpA auxotrophs is somewhat
4
E. .J. MI’K(:C)lA
surprising since wild-type trpA has ten codons that could become UGA by a single base-change (Nichols & Yanofsky, 1979). Nevertheless. it seemed to us that, as long as UGA mutants of trp.4 are auxotrophic. we should be able to select for the conversion to UGA of existing trpA mutations (related to UGA by a single basechange) in the presence of a UGA suppressor. Such a suppressor, of course, would have to insert an amino acid that is functional at the mutant position of the protein. In the past, however, the well-characterized UGA suppressors were known either to insert tryptophan or to be derived from tryptophan tRNA (Chan & Garen, 1970: Hirsh, 1971 ; Ghan rt al., 1971 : Soll, 1974). Since there is no tryptophan residue in the wild-type TSase alpha chain, it is possible that the occurrence of that amino acid at any one of the ten positions rtprrsented by UGA-related codons results in a non-functional alpha chain (two positions have leucine. two serine, three cysteine, one arginine. and two glycine). If such were the case, it would not be possible to obtain or identify a U(:A muta,nt’ wit-h t,hose suppressors. Recently, however. UGA suppressors were obtained from a glycine tRNA (Murgola & Childress; 1980). In order to obtain trpA(~~GA211). we constructed the following strain: glyT(SuUGA/G) AtonB-trp/F’trpd (lTAA2I 1). it contains a glyT-derived suppressor of IT(:A and IJGG that was previously designated SP-11 (Murgola & Childress. 1980). The ochre mutation trp;l (FAA21 1) occurs on the Fredericq episome, which covers a chromosomal deletion from tonB to (at least) trpB. Since the enzymatic behavior is known for alpha chains containing each of 15 amino acids at position 211 (Murgola & Yanofsky. 1974c), one can predict precisely the nature of all prototrophs derivable from most trp.4 mutations at that position. Table 1 indicates the five possible single-step mutations in a frp-q(UAA211) strain that contains g/yT(SuUGA/G). In fact,, class B-2 was not considered likely to occur since the most frequent,ly occurring spontaneous ochre suppressors carry amino acids that. when inserted at position 211 of the alpha chain, yield a non-functional protein. Thus four classes remained. We were able to screen directly, however. for class B-3 as described in Materials and Methods. In that, fashion, several suppressed UGA were identified. After single-colony isolation, several mutants of trp.4 trp=l (UGA21 I)-containing episomes were transferred to a specifically marked. suppressor-free ton B-trp deletion strain.
Possihilitirs
for
single
step
prototrophic
trpAB/F’trpA A. trpA
dwivatiws of glyT(Su (I:=1 .d 21 I)
revertants
1. gZy?‘(SuUGA/G) 2. gZyT(SuUGA/G)
H. Suppressed
trpA
dtonR-trpAB/F’trpil(UCAIll) dtonB-trpAR/F’tr~(~~rU~ll)
mutants
I. glyT(SuUAA) dtonB-trpiiB/F’trpA(I.AA-“I 1) 2. SuU,4A gZyT(SuUGA/G) dtonB~tr~AB/F’trpA(ITAA~ll) 3. gZyT(SuUGA/G) dtonR-trpAB/F’tr~A(~GAZll)
UU=l/C?)
Aton-
UGG-SPECIFIC
(b) Suppressor
selection
for
6
SUPPRESSORS
trpA(
UGA234)
and
trpA(
UGG234)
Glycine is also the wild-type amino acid at position 234 of the alpha chain. One of the missense mutations affecting that position involves replacement of glycine by cysteine. It was formerly designated trpA78 and is due to the codon change of GGU to UGU (Yanofsky et al., 1966: Nichols & Yanofsky, 1979). We now refer to this mutant as trpA(UGU234). The existence of this mutant was particularly fortunate for us in that, in the presence of a UGA/G suppressor, it could theoretically undergo single-step mutations to both UGA and UGG. Until the present study, only at position 211 did a UGG mutation exist that was useful for selection of new suppressors (Murgola & Yanofsky, 1974c; Murgola & Childress, 1980). Indirect evidence, however, suggested that tryptophan is not functional at position 234. The parent strain constructed in order to obtain the new mutations at position 234 was gZyT(SuUGA/G) dtonBtrpAB/F’trpA(UGU234). Since not as much is known about amino acid replacements at positon 234 as at position 211: we cannot make a list of all possible single-step prototrophic derivatives of the parent strain, as we did in the previous section for position 211 (Table 1). But when codon-specific suppressors exist, that lack of information is no handicap. Consequently, we screened prototrophic derivatives of trpA(UGU234) as described in Materials and Methods, with one exception. In the replica-plate matings to transfer out the episomes. between the first mating (into a suppressorfree st’rain) and the final mating (into the UGA/G-suppressor strain) we included a mating into a strain containing a gZyEderived UGG suppressor (Murgola $ (‘hildress, 1980). glyU codes for a glycine tRNA that responds to GGG but not (EA. The rGG suppressor derived from it does not suppress a phage T4 UGA mutation (one that is suppressed by other glycine tRNA UGG suppressors), and so appears to be UGG-specific, as expected. In the screening, F’trpA(UGG234) was identified by yielding Trp+ exconjugants in both the second and third matings. whereas F’trpA((UGA234) did so only in the third mating. After single-colony isolation, isolates of each type were verified by individual matings.
(c) Growth
of trpA mutants, suppressed on indolef5-methyl-nL-tryptophun
and unsuppressed, medium
Missense mutants of trpA make a complete, although enzymatically inactive, TSase alpha chain. Such mutants produce an alpha chain that can activate the TSase beta subunit in the conversion of indole to tryptophan. They are able, consequently, to grow on glucose minimal medium containing a low concentration (15 pg/ml) of indole and a high concentration (50 pg/ml) of 5-methyl-nLtryptophan (Ind-5MT). In the presence of 5MT, which acts as a corepressor, the trp operon is fully repressed. In this condition, a low amount of the beta chain is made, but it is insufficient to allow the bacterium to grow on 1.5 pg of indole per ml unless the bacterium also produces a complete alpha chain that can activate whatever amount of beta chain is made. Consequently, since nonsense trpA mutants do not make a complete alpha chain that can activate the beta chain, they cannot grow on Ind-5MT.
6
E. I. MURGOLA
Such reasoning could lead one to expect that, in the presence of a nonsense suppressor of the appropriate codon specificity, a given trpA nonsense mutant would grow on Ind-5MT. We found this to be the case (Murgola & Jones, 1978) in the suppression of trpA(UAG211) by the amber suppressors supD and ,supF, which insert serine and tyrosine, respectively. However, when we tested trpA(UAA211) and trpA(UAG211) in the presence of an ochre suppressor derivative of supD, the strains did not grow on Ind-5MT (Murgola & Jones, 1978). We considered this result reasonable because ochre suppressors are generally much less efficient than amber suppressors. We suggested that any trpA nonsense mutant in the presence of a corresponding low-efficiency suppressor does not produce enough complete alpha chains in the repressed state of the trp operon (i.e. in the presence of 5MT) to activate the beta chains sufficiently to allow growth on Ind-5MT. The experimental basis for the discussion in the previous two paragraphs involved only amber and ochre mutants because until the present study no UGA mutation in trpA had been identified. Consequently, we were interested in determining whether the two trpA UGA mutants can grow on Ind-SMT, in the presence or absence of the UGA suppressor SP-11 and others recently isolated by US. As determined either by replica-plating of patches or by streak outs from cell suspensions, neither trpA(UGA211) nor trpA(UGA234) was able to grow on Ind5MT. As expected, however, trpA(UGG234), being a missense mutant, was able to grow on that medium. Growth of the UGA mutants in the presence of various UGA suppressors is reported in the next section. (d) Suppressor
response
to UCA
and UGG at two positions
in trpA
Recently (Murgola & Childress, 1980), we isolated several classes of UGG suppressors using the mutant trpA(UGG211). Some did not suppress a certain UGA mutant of phage T4 and so appeared to represent UGG-specific suppressors, that is, ones that do not exhibit “wobble” with regard to the third position of the codon. Especially interesting were those derived from tRNAs that do exhibit such wobble, namely gZyl’55 and gZyT (see Table 2). To test this loss of wobble using bacterial TABLE tRlVA Gene symbol
genes from
which supn
UCX
2
suppressors
were obtained
C&V55
mutationally
SYT
SlYU
Codon recognized (5’ 43’1
UAG
Anticodon (3’+5’)
-a
GGA.
GGG
CCl’b
a supD tRNA is derived from a minor swine tRNA ant.irodon (D. Steege, personal communication). b Squires & Carbon (1971). ’ Roberts & Carbon (1975); firepresents an unidentified the gZyT tRNA molecules isolated from B. roli veils. d Hill ef al. (1973).
GGA,
GGG
GGG
C&F
by a single
derivative
ccc*
base change
of U that
in the middle
is present
of the
in a portion
of
glyV5P
SP-I. SP-3, SP-5, SP-6,
+
+ +
+
-
-
+
-
+
+
+e
Mind
ITGG2 11
-
Suppression of a phage T4 C’GA mutantC
-
+
-
+
-
-
-e
Min
EGA21
-
-
-
+
-
Tnd-5MTd
1
+
+
+
+
+
-
Min
UGG”34
-
-
-
a For origin and nomenclature, see Materials and Methods. bSee Table 2 for codon specificities and anticodons. ’ From Murgola & Childress (1980). dMin, glucose minimal medium; Ind-5MT, Min plus indole(l.5 pg/ml) and 5.methyl-DI.-tryptophan (50 &ml). e + , growth, and -, no growth, on Min or Ind-BMT after 5 days of incubation at 37°C. ‘This suppressor arose in a supD-containing strain, is linked to his, retains supD activity. and so far has not been separated B A mutant form of gZyV, which codes for a GGU/C-reading glycine tKNA.
glyV55
c&d’
SP-13
SP-9,
SP-14
sW”
EMS-B, EMS-e, EMS-7, EMS-& EMS-I), SP-11, SP-18, SP-19, SP-20
SP-4, SP-7
sly CT
SP-15
supD
?
EMS-2 EMS-4’
Parental tRNA geneb
SP-12’
EMS-l EMS-31
Suppressors examined”
from
supD.
-
-
genetically
-
-
-
In&5MT
-
+
-
Min
UGA234
8
E. ,I. MUR(:OLA
mutations, we obtained two homotopic pairs of UGA and UGG mutations, at TSase alpha chain positions 211 and 234, as described in the first two sections of Results. We then constructed tonB-trp deletion strains containing representatives of each of several categories of those suppressors. We emphasize that each suppressor was first removed, by transduction, from its original mutagenized or selective genetic background. Through conjugation, Fredericq episomes carrying the four trpA mutations (UGA and UGG, at positions 211 and 234) were introduced into each suppressor-containing trp deletion strain by selecting for growth on glucose minimal medium plus indole (trpB). Finally. suppression was determined by testing the exconjugants for growth on glucose minimal medium (Min) and on Min plus low (1.5 pg/ml) indole and high (50 pg/ml) 5-methyl-uL-tryptophan (Ind-5MT). The results are shown in Table 3. Neither SP-12 nor any of the suppressors derived from supD or glyU exhibit suppression of either UGA mutation. Also, whereas SP-15 suppresses trpL4(UGG234), SP-12 and EMS-l, EMS-Z, EMS-3, and EMS-4 do not. trpA(UGA211) is suppressed by six gZyV55-derived suppressors and nine glyT-derived ones. Those nine gEyT suppressors, however, are unique in two respects: (1) only they suppress trpA(UGA234) on Min; and (2) only they allow trpA(UGA211) to grow on Ind-5MT, although they do not do so for trpA(UGA234). All st.rains containing trpA(UGG211) or trpA(UGG234) grow on Ind$MT, even in t*he absence of any suppressor (not shown in Table 3). After introduction of each episome into each suppressor-containing trp deletion strain, all of the resulting strains were not only tested for growth on Min and Ind5MT but also examined as follows. To show the presence of the proper trpA mutation, the episome was transferred by mating into appropriate suppressor strains. To verify the functional presence of the correct suppressor two things were done. (1) For those strains that did not’ grow on Min, F’trp.4 (UGG211) was mated in and the exconjugants tested for growth on Min. (2) Phage Pl lysates were made on all strains and then tested for the correct suppressor by transductional linkage to the proper marker (purrl, metB. @A and his).
4. Discussion In this paper we have reported (1) the isolation of trpA mutants representing UGA at TSase alpha chain positions 211 and 234, as well as UGG at position 234; and (2) the use of these new mutants to test the response to them of several classes of UGG suppressors. Until now, no UGA nonsense mutations have been found in trpA or verified in any other trp operon gene in E. coli. The recent isolation, however, of a UGA suppressor derived from a glycine tRNA (Murgola & Childress, 1980) provided the opportunity to look for UGA at alpha chain positions where glycine is the wild-type amino acid. At position 211 the glycine codon is GGA, one of the ten trpA codons related to UGA by a single base change. The glycine codon at 234 is GGU and would not have yielded a single-step UGA mutation. Using the suppressor selection method, we readily obtained UGA mutations at both positions.
UGG-SPECIFIC
SUPPRESSORS
9
Despite previous observations concerning UGA mutations in E. coli and Salmon& (e.g. see Roth, 1970), the new mutants trpA(UGA211) and trpA (UGA234) are not leaky ; they are cleanly TrpThis is true in strains that are not streptomycin resistant and have no known ribosomal mutations (that is. mutations that might result in restriction of some hypothetical low-level UGAsuppressing activity). These mutants also do not grow on Ind-5MT medium (see Materials and Methods) as is true of all nonsense mutants (UAA and UAG) of trpA examined thus far (Murgola & Yanofsky, 1974c; Murgola & Jones, 1978). The failure of trpA(UGA211) to grow on that medium in the presence of six gZyVn’5derived UGA suppressors indicates that the efficiency of those suppressors is very low compared with the nine g&T UGA suppressors and with the highly efficient amber suppressor supD (Murgola & Jones, 1978). In Results section (a), above, we discussed the possibility that previous studies may not have detected UGA mutations due to the amino acid specificity of the existing suppressors. Among the trp operon mutants obtained by Yanofsky and coworkers, there are some that were determined to be due to chain-terminating mutations but were not suppressed by several different nonsense suppressors (see. e.g. Yanofsky et al., 1971). But generally only one UGA suppressor was employed, a tryptophan-inserting one. Therefore, some of those unidentified-chain-terminators may be UGA mutants. Consequently, testing them with one or several of our new ITGA suppressors might identify a variety of useful and perhaps very interesting ITUA mutations. All three nonsense codons now exist at position 2 11, and the presence of UGA at position 234 allows us to select for UAA and UAG in t,he presence of ochre and amber-specific suppressors, respectively. Furthermore, since the UGA selection worked in these two instances it should be possible to obtain UGA, as well as UAA and UAG, at other positions in trpA , regardless of whether they are presently sites of nonsense or missense mutations. Having this homotopic triad of nonsense codons at, many positions within the same well-characterized gene will make possible the desirable extension of studies begun (Feinstein & Altman, 1977,1978; Bossi & Roth, 1980: Fluck & Epstein, 1980) on the effects of mRNA context and tRNA structure on the process of translation of the genetic code. Some years ago, an indication of the non-functionality of alpha chains carrying tryptophan or serine at position 234 was provided by indirect evidence, namely the absence of tryptophan or serine-substituted prototrophic revertants of trpA(UGU234), formerly designated trpA78 (Yanofsky et al., 1966). In the present work, direct evidence for the behavior of tryptophan was obtained by our ability to generate trpA (UGG234) as an auxotroph. Furthermore (data not presented), trp=l (UGA234) is not suppressed to Trp+ by a tryptophan-inserting UGA suppressor (Sambrook et al., 1967). The same is true of trpA4(UGA21 l), but that was expected on the basis of the known amino acid requirements of alpha chain position 211 (Murgola B Yanofsky, 1974c). Less conclusive, however, is the result concerning serine, namely that suppression of trpA(UGG234) to prototrophy was not exhibited by the four UGG suppressors derived from the serine-inserting amber suppressor supD. The force of the conclusion is weakened by the fact, that we cannot’ be certain that the UGG-reading forms of that tRNA still insert serine.
10
E. .J. MCRGOLA
Furthermore, the failure to suppress trpA(UGG234) as opposed to trpil(UGG211) could be due to the difference of the nucleotide sequence “context” of their mRNA codons (Nichols & Yanofsky, 1979) rather than to the nature of the amino acid inserted. Once trpA (UAG234) is obtained, however, it should be possible to provide definitive evidence by demonstrating that supD itself, known to insert serine, relieves chain termination at that trprl nonsense codon and produces a complete but’ enzymatically inactive alpha chain (Murgola & Yanofsky, 1974c; Murgola $ Jones, 1978). The inability of the supD-derived UGG suppressors to respond to UGA is not surprising since supD tRNA reads UAG but not UAA (see Table 2). A similar comment can be made about gly U suppressor SP-15, which is derived from a glycine tRNA that reads GGG but not GGA (Carbon et al., 1970; Murgola & Pagel, 1980). Since the identity of the his-linked suppressor SP-12 is unknown, no prediction was possible regarding its response to UGA. As it turns out, it does not suppress either UGA mutation or trpA(UGG234). SP-12 could be derived from a cysteine tRNA gene that is located in the supD region of the chromosome (Ikemura & Ozeki, 1977). It is known that the occurrence of cysteine at position 234 yields a non-functional alpha chain (Yanofsky et al., 1966). Finally, another category that was understandably anticipated comprises SP-11 and eight other glyT suppressors that respond to all three UGA mutations. Since they are derived from a species that reads A or G in the third position of the codon, we expected that such UGG suppressors would also suppress UGA. Surprising results were obtained, however, with the remaining suppressors (two derived from glyT, and seven from glyV%), and three considerations arising from their behavior implicate aspects of tRNA structure other than the anticodon in the specificity of codon recognition. First, since glyT and glyV55 each gave rise to at least two classes of UGG suppressors, at least two kinds of single nucleotide change must be involved in each case. Therefore, at least one type of change must be one that is not predictable according to classical codon-anticodon hydrogen-bonding rules. Second, although the tRNAs coded by glyT and glyV55 respond to GGA in addition to GGG, two suppressors derived from the former and one from the latter fail to suppress the two bacterial UGA mutations in addition to the phage T4 mutation. The fact that all three suppress homotopic UGG codons at two positions in trpA (211 and 234) shows that misacylation is not involved in the failure to suppress the bacterial UGA mutations. It must be kept in mind that the conversion of any glycine tRNA to a UGG suppressor involves primarily a change in response to the first position of the codon. But’ in the case of the UGA-restrictive suppressors. any such change, whether it is in the anticodon or elsewhere in the molecule, must also effect the third-position response. Consequently, an effect on tRNA conformation is indicated. We consider it unlikely that these suppressors are due to two nucleotide changes having occurred in one mutational event. First, the frequency of their occurrence is high (Murgola & Childress, 1980; and unpublished data). Second, only one type of single-step translational suppressor has been shown to be due to two nucleotide changes, and that kind occurs at a very low frequency and in response to nitrosoguanidine or ultraviolet irradiation (Coleman et al., 1980). We cannot rule out. however, the possible involvement of a cryptic mutation in a
UGG-SPECIFIC
SUPPRESSORS
11
T-ABLE 4 Messenger
RIvA
Alpha
position
chain
mKNA
codons
Amino
acids
t Nichols
codon contexts? the tryptophun 209 UUG Leu
& Yanofsky
of UGA mutations corresponding to two position,s synthetase alpha chain of E. coli 210
211
CAG
I’GA
Gln
.- -
212 UUC Phe
313.. GGU Gly
232 9tW Ile
233 UCI’ Ser
234
235
LTGA UCG --~
Ser
in
236.. GCC Ala...
(1979)
closely linked gene that codes for a tRNA-related molecule (for example, a tRNAmodification enzyme). The third consideration concerning tRNA structure and codon recognition is mRNA context effects. Six of the suppressors derived from gZyV55 suppress trpA(UGA211) but not trpA(UGA234), although they all suppress UGG at both positions. Also, in the presence of the nine gZyT UGA suppressors, trpA(UGA211) is suppressed sufficiently to grow on Ind-5MT, but trpA(UGA234) is not. The nucleotide sequences in the neighborhood of UGA211 and UGA234 are clearly different (Nichols & Yanofsky, 1979; and Table 4). Nevertheless, neither region displays features that bear any obvious relation to previous findings concerning suppression of UAA or UAG in different contexts (Feinstein & Altman: 1977,197s: Bossi & Roth, 1980). Such differential context-dependent responses to a given codon by one and the same tRNA, observed now for all three nonsense codons. cannot be due simply to codon-anticodon hydrogen bonding. The influence of the. nucleotides adjacent to the codon could be by way of an effect on mRNA secondary structure (Grosjean et aE., 1978; Grosjean & Chantrenne, 1980). Alternatively, it may involve differential interactions of the suppressor tRNA with those nucleotides or with the ribosome, an adjacent tRNA, or release factors. Finally, the effect might be manifested after peptide bond formation in the stability of the peptidyl-tRNA (Caplan & Menninger, 1979), or in the generation of shifts in reading frame (Atkins et al., 1979; Kurland, 1979). We have not yet determined, by enzyme assays, the efficiencies of suppression of trpA(UGG211) and trpA(UGG234) by any of the suppressors. Nevertheless, those that display context-dependent ITGA suppression show no gross difference in suppression of UGG at the two positions as judged by rates of growth in liquid medium and colony size on solid medium (data not presented). This fact suggests that in these cases, the context effects relate more to factors involved in chain termination than to parameters such as tRh’A-tRNA interaction. It will be interesting to investigate whether any missense suppressors display context effects in their response to sense codons. The isolation of glyT- and gZyV55-suppressor tRNAs for nucleotide sequence analysis is underway. In the primary-structure analysis we are concerned first with the location and nature of the mutationally determined nucleotide change. Of special interest is the anticipation that some suppressors might be due to mutations quite distant from the anticodon, as in the case of the well-studied tryptophan
12
E. ,I. MUKGOLA
tRNA UGA suppressor (Hirsh, 1971). But we must also ask whether any change in base modification has occurred. Specifically. is the adenine that is adjacent to the anticodon on the 3’ side modified with an isopentenyl moiety? If so. to what extent? Is the modified uracil in the 5’ position of the anticodon of gEyT tRNA (see Table 2) under-modified or modified differently? Has the uracil in the 5’ position of the anticodon of gZyV55 tRNA, or any other normally unmodified base in either species, become modified as a result of the primary structure change? Particularly intriguing is the question of the nature of the changes that restrict third position wobble (this paper) or allow.gZyT UGA suppressors to exhibit an unorthodox wobble with regard to the first position of a codon (Murgola & Pagel, 1980). Once we have t,he answers to these questions. we may be able to make specific correlations between tRNA structure and the specificity of codon recognition. In any event, it is clear in general, from the present study and previous work of others, that parts of the tRNA structure other than the primary anticodon sequence must be involved in the process of codon recognition, whether it be by way of nucleotide modifications or conformational changes of the tRNA molecule (see Introduction, and also Mijller et al., 1979). In the light of those studies and recent, ones on mitochondrial tRNAs (Barrel1 et al., 1979,198O ; Bonitz et aZ., 1980 : Eperon et a,l., 1980; Heckman rt al.. 1980). it has become necessary to rethink the “rules” for codon recognition by transfer RNAs. One may wonder, however, how likely it is that any universal rules can be formulated that will apply uniformly to all tRNAs. For excellent technical assistance with different portions of this work, 1 am most grateful t,o Frances Pagel, Kathryn Hijazi and Patrick Condreay. I also thank Charles Yanofsky for his comments on the manuscript and Walter Pagel, Janet Naquin and Janie Finch for assistance in its preparation. Portions of this work were supported by grants from the American Cancer Society (NP-167) and the U.S. Public Health Service (GM 21499, from the National Institute of General Medical Sciences). The author is the recipient of U.S. Public Health Service research career development award GM 00047 from The National Institute of General Medical Sciences.
REFERENCES Atkins, J. F., Gesteland, R. F., Reid, B. R. & Anderson, C. W. (1979). CeZl, 18, 1119-1131. Barrell, B. G., Bankier, A. T. & Drouin, J. (1979). Nature (London), 282, 189-194. Barrell, B. G., Anderson, S., Bankier, A. T., de Bruijn, M. H. L., Chen, E., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R. & Young, I. G. (1980). Proc. ,Vat. Acud. Sci., U.S.A. 77,3164-3166. Berger, H. & Yanofsky, C. (1967). Science, 156, 394-397. Bonitz, S. G., Berlani, R., Coruzzi, G., Li, M., Macino, G., Nobrega, F. G., Nobrega, M. P., Thalenfeld, B. E. & Tzagoloff, A. (1980). Proc. Nat. Acad. Sci., U.S.A. 7’7, 3167-3170. Bossi, L. & Roth, J. R. (1980). A’ature (London), 286, 123-127. Buckingham, R. H. & Kurland, C. G. (1977). Proc. IVat. Acud. Sci., U.S.A. 74, 5496-5498. Caplan, A. B. & Menninger, J. R. (1979). J. Mol. Biol. 134, 621-637. Carbon, J., Squires, C. & Hill, C. W. (1970). J. Mol. Biol. 52, 571-584. Chan, T. & Garen, A. (1970). J. Mol. Biol. 49, 231-234. Chan, T., Webster, R. E. & Zinder, N. D. (1971). J. Mol. BioE. 56, 101-116.
CGG-SPECIFIC
SI:PPKESSOR~
I3
Coleman. R. D., Dunst, R. W. & Hill, C. W. (1980). Mol. (:en. Drnet. 177, 213-222. Crick, F. H. C. (1966). J. Mol. Biol. 19, 548-555. Eperon. 1. C.. Anderson, S. & Nierlich, D. P. (1980). Suture (London). 286, 46&467. Feinstein. S. I. &, Altman, S. (1977). J. Mot. Biol. 112, 453-470. Feinstein, S. I. & Altman, S. (1978). Urn&s, 88, 201-219. Fluck, M M. & Epstein, R. H. (1980). Mol. Gen. Genet 177. 615627. Fredericq, P. (1969). In Bacterial Plasmids and Episomrs (Wolstenhome. (:. E. W. 8r O’Connor, M., eds), pp. 1633174, Little, Brown and Co.. Boston. (Goldman. E.. Holmes, W. M. & Hatfield. G. W. (1979). J. Mol. Biol. 129, 567-585. (irosjean. H. $ Chantrenne, H. (1980). In Chemical Recognitiorc in Biology (Chapeville, F. 8i Haenni, A.-L.. eds), pp. 3477367, Springer-Verlag, Berlin. (irosjean. H. J.. de Henau. S. & Crothers, D. M. (1978). Proc. Sat. =Icad. Sci.. 1I.S.d. 75. 610-614. Hadley, K. H. & Murgola, E. J. (1978). (‘urr. ~~icrohiol. 1, 99-103. Heckman. ,J. E., Sarnoff, J., Alzner-DeWeerd, Tin. S. & RajBhandary. U. L. (1980). Proc. .\rut. .jcad. Sk., U.S.A. 77: 315993163. Hill. C. WT., (‘ombriat.o, G.. Steinhart. W.. Riddle. D. L. & Carbon, J. (1973). J. Biol. Chrm. 248. 42.X-4262, Hirsh, D. (1971). J. Mol. Biol. 58, 439-458. Ikemura, T. & Ozeki, H. (1977). J. Mol. Biol. 117, 419-446. Kurland, C. G. (1977). dnnu. Rev. Biochem. 46, 173-200. Kurland, C. G. (1979). In AVonsense Mutations and tR,VA Suppressors (Celis, J. E. & Smith. ,J. D.. eds). pp. 977108, Academic Press, New York and London. Kurland, C. C:., Rigler, R.. Ehrenberg, M. & Blomberg, C. (1975). Proc. ivat. Acud. Sei.. l’.S..1. 72, 424884251. Mitra. S. K., Lustig, F., Akesson, B.. Lagerkvist, U. & Strid. L. (1977). J. Biol. Chem. 252, 471-478. Miiller. A., Mandershied, U., Lipecky, R., Bertram, S., Schmitt, M. & Gassen, H. G. (1979). In 7kxnsfer RSA: Structure, Properties, and Recognition (Schimmel, P. R., Still, D. & Abelson, J. N., eds), pp. 4599471, Cold Spring Harbor Laboratory, New York. Murgola. E. ,J. & Bryant, J. E. (19801. J. Bucteriol. 142, 131-137. Murgola. E. ,J. & Childress, J. R. (1980). J. Bacterial. 143, 285-292. Murgola. E. ,J. & Jones, C. I. (1978). Mol. Gen. Genet. 159, 179184. Murgola. E. ,J. &, Pagel, F. T. (1980). J. Mol. Biol. 138, 833-844. Murgola. E. ,J. & Yanofsky, C. (1974a). J. Bacterial. 117, 439-443. Murgola. E. J. & Yanofsky, C. (19746). J. Bacterial. 117, 444-448. Murgola. E. ,J. 8: Tanofsky, C. (1974c). J. Mol. BioZ. 86, 775-784. Murgofa. E. ,I., Prather, N. E. & Hadley, K. H. (1978). J. Bacterial. 134, 801-807. Nichols? B. P. & Yanofsky, C. (1979). Proc. Sat. ilcad. Sci., I!.S.=l. 76, 52445248. Pope. W. T., Brown. A. & Reeves. R. H. (1978). Xucl. Acids Res. 5. 104-1057. Reeves. R. H. & Roth, J. R. (1975). J. Bacterial. 124, 332-340. Roberts. ,J. W. & Carbon, J. (1975). J. Biol. Chem. 250, 553&5541, Roth. J. K. (1970). J. Bacterial. 102. 467-475. Sambrook, *I. F., Fan, D. P. & Brenner, S. (1967). ,Vature (London), 214, 452-453. Smith. ,J. D. (1979). In Sorrsense Mutations and tRIVA Suppressors (Celis, J. E. & Smit.h. ,J. D.. eds), pp. 109125, $cademic Press, New York and London. Sol], L. (1974). J. Mol. Biol. 86, 2333243. Squires. C. & Carbon, J. (1971). Nature Sew Biol. 233, 274-277. Steege. D. A. & Still, D. G. (1979). In Biological Regulation and Development (Goldberger, R. F.. ed.), vol. 1, pp. 4333485, Plenum Publishing Corp., New York, Vaeher. J. & Buckingham. R. H. (1979). J. Mol. Biol. 129, 287-294. Yanofsky. C.. Ito, J. & Horn, V. (1966). CoZd Spring Harbor Symp. f&ant. Biot. 31, 151-162. Yanofsky, C., Berger, H. & Brammar, W. J. (1969). Proc. XII Int. Coqr. Genet. 3, 155-165. Tanofsky. (‘.. Horn. V., Bonner, M. & Stasiowski. 6. (1971). C:enetics, 69, 409433.