The effect of an Escherichia coli regulatory mutation on transfer RNA structure

J. Mol. Biol. (1979) 135, 111-126

The Effect of an Escherichia cdi Regulatory Mutation on Transfer RNA Structure STEPHEN

P. EISENBERQ,

MICHAEL

YARUS AND LARRY SOLL

Department of Molecular, Cellular and Developmental Biology University of Colorado Boulder, Co.!. 80309, U.X.A. (Received I2 March 1979, and in revised form 31 July 1979) The trpX mutation in Eecherichia coli reduces trp operon attenuation in strains tRNA TrP. The trpX- phenotype carrying wild-type is alleviated (attenuation is restored) in UGA-suppressor tRNA T’*-carrying strains (Yanofsky & Soll, 1977). The tRNA from various trpXstrains was characterized biochemically. Sequence analyses of wild-type tRNATPr and UGA suppressor tRNATPr, both derived from trpX- strains, reveal an unmodified A in the position (adjacent to the anticodon) normally occupied by the hypermodified base ms2i6A. In addition, several tRNAs from trpXcells were characterized by RPC-5 normally having ms2i6A column chromatography. We find that only tRNAs exhibit altered elution profiles when compared to the homologous tRNAs from trpX- cells. Introduction of the UGA suppressor into trpX- cells does not restore normal chromatographic behavior. These results suggest that the trpX gene product is necessary for the synthesis of ms2isA. Thus, we propose that r&A (for the first gene involved in wa.sVA synthesis) replaces the trpX designation. The results reported here are discussed with regard to a model proposed by Lee & Yanofsky (1977) in which efficient translation of the tandem trp codons in the leader sequence RNA is required for normal attenuation of the trp operon.

1. Introduction of the trp operon

Transcription

is primarily

regulated

at two sites. At the operator

site

by interaction with an aporepressor-Ltryptophan complex (reviewed by Yanofsky, 1976). The operon is also regulated by a mechanism which functions in series with the repressor-operator system (Bertrand et al., 1975). This system controls expression by terminating transcription at a site called the attenuator, which has been located (by mapping with deletion mutants) in the leader sequence, between the operator and the first structural gene for tryptophan synthesis (Jackson & Yanofsky, 1973). The termination (or attenuation) process is clearly dependent on the proper functioning of tRNATrp and tryptophanyl-tRNA synthetase (trpRS) and is apparently dependent on the level of charged tRNATrP in vivo (Morse & Morse, 1976; Yanofsky & Soll, 1977). The sequence of nucleotides in the leader sequence of the trp operon contains an AUG (initiator) codon (which can function as a ribosome binding site in vitro ; Platt et al., 1976), two tandem UGG (tryptophan) codons, and a UGA (termination) codon, all in the same phase (Squires et al., 1976; Lee et aZ., 1978). If the RNA transcribed from this region were to be translated normally, a polypeptide 14 amino acid residues initiation

of

transcription

is

controlled

111 0022-2836/79/330111-16

$02.00/O

0 1979 Academic

Press Inc. (London)

Ltd.

112

S. I’.

EISENBERG,

hl. YAKI:X

AN I) 1,. SOT,I,

in length would result, and t’he two tryptophan residues would be at positions 10 and 11 (Lee & Yanofsky, 1977). Lee & Yanofsky (1977) propose two alternative hairpin loops in the leader region RNA. One possible structure goes from nucleotidr 74 to nucleotide 119: and contains a 12 base-pair stem and a non base-paired 22 nucleotide loop (see Fig. 1). The other structure includes nucleotides from residue 114 to 134 with a G ; C-rich eight basepair stem and a four base-pair loop (Fig. I). This second hairpin loop could he a part

FIG. 1. Alternative hairpin loops in leader seyuencu RNA. The nuclcotide sequence 1s from Lee & Yanofsky (1977). (a) AC = -12 kcal/mol. (b) A(: -~: --20 kcal/mol. See text. Hyphens omitted for clarity from all Figs.

of the termination signal (preceding the short stretch of uridylates) which acts to retard the elongating RNA polymerase allowing it to terminate and dissociate from the DNA (Pribnow, 1978; Stauffer et al., 1978). The two hairpin structures along with their respective calculated stabilities (Tinoco et al., 1973; Borer et al, 1974) are shown in Figure I. These ideas regarding the sequence and secondary structure of the leader region RNA, along with the involvement of charged tRNATr* in the attenuation process, were combined by Lee & Yanofsky (1977) into a model for attenuation in which translation of the leader RNA occurs with the ribosome following closely behind t,he . plentiful supply translation can RNA polymerase molecule. If charged tRNATr* is . in occur through the trp codons to the UGA nonsense codon (nucleotides nos 69 to 71). The presence of the ribosome at that position should prevent the formation of the hairpin loop in the region from nucleotide 70 to 119, since the ribosome probably covers about 25 bases or at least 10 bases on either side of the A and P site codons (Steitz, 1978). This allows the terminator loop to form (114 to 134) a(nd attenuation to

frpX

MUTATION

AFFECTS

tRNA

113

MODIFICATION

starvation the transIf charged tRNATrP is in short supply due to tryptophan lating ribosome should become stalled at the UGG codons, the 74 to 119 hairpin would be allowed to form and formation of the terminator loop would be prevented. Transcription of the remainder of the operon occurs in this situation. Mutants of Escherichia coli have been isolated in which the structure of tRNATrP site is altered and the cell’s ability to regulate trp operon expression at the attenuator is impaired (Yanofsky & Soll, 1977). One of these, trpX, is particularly interesting because it affects the chromatographic behavior of tRNATrP but does not map within the structural gene for that tRNA. Strains carrying the trpX- allele have higher than normal levels of trp operon expression due to less efficient attenuation. In this paper strains to that of wild-type we compare the sequence of tRNA Trp isolated from trpX(trpX + derived) tRNATrp. In addition we have examined tRNA from a double mutant (Hirsh, 1971). The UGA nonsense supressstrain carrying both trpXand trpT-su&A ing allele of trpT restores normal attenuation even in the presence of the trpX mutation (Yanofsky & Soll, 1977). These results are discussed in terms of the translation model of Lee & Yanofsky (1977). occw.

2. Materials and Methods (a) Bacterial LS1074:

W3110

trpR-

trpT+

and derivations

strains

trpX + la&,,,,

(Korn

& Yanofsky,

1976; Yanofsky

&

Soil, 1977). LS1075: W3110

trpRtrpT+ trpXZacZ,,,, (Yanofsky & Soll, 1977). Phage PI transduction of LS1075 trpRtrpTsu&, trpXZacZ,,,,. u7it.h P 1 (trp TN&, (Yanofsky & Soll, 1977). Pl transduction of LX1074 with Pl LS1273: W3110 trpRtrpT+ trpXTnlO ZacZ,,,,. grown on strain having T%lO integrated near trpX(G. Zurawski & C. Yanofsky, personal communication). LS1076:

W3110

(b) Bacterial The growth medium for [32P]orthophosphate Cells grown in either 25 pg L-tryptophan/ml below) chromatography

growth

media

of Garen & Levinthal (1960) with 25 fig L-tryptophan/ml w&9 used (carrier-free, New England Nuclear) labeling of cellular RNA. LB medium (Miller, 1972) or Vogel-Bonner (1956) medium with were the source of the tRNA used in the RPC-5 (see section (e), experiments. The tRNA from both media gave similar results. (c) Isolation

of tRNA

[32P]tRNA was extracted with phenol from cells by the method of Ikemura & Dahlberg ( 1973). Unlabeled tRNA was extracted by the method of von Ehrenstein (1967). Purification of [32P]tRNATr* was by 2-dimensional polyacrylamide gel electrophoresis in a system described by Ikemura & Nomura (1977) (0.1% TEMEDt was substituted for 0.4% 3dimethylaminopropionitrile). The gel dimensions were 16 cm x 20 cm. Electrophoresis in the 1st dimension was at 20 mA for 3 h, in the 2nd dimension, 350 V for 50 h. Acrylamide was purchased from Kodak. The procedure for autoradiography and elution of tRNA from gels was similar to the method described by Ikemura & Dahlberg (1973). The gel elution solution was 0.6 M-NaCl, 0.5% phenol, and 20 pg yeast RNA/ml. The gel suspension was vortexed several times every few hours during incubation at 21°C for 12 h. The entire suspension was then filtered through silicone-coated glass wool into a small centrifuge tube, then precipitated by the addition of 0.1 vol. 5 M-NaCl and 2 vol. ethanol. t Abbreviations mononucleotides

used: TEMED, N,N,N’,iV’-tet~ramethylethylene of adenosine, cytidine, guano&e and uridine.

diamine;

A,

C, G and

U,

114

S. P. EISENBERG,

&!I. YARUS

AND

1,. SOLL

(d) Sequence a?~alysis T, (Sankyo) and pancreatic (Worthington) RNase fingerprint,ing wa,s performed as bl Griffin (1971). After nucleotides were transferred ontao polyrthylono imine (PEI)-cellulosc~ plates, the plates were carefully washed with ZOO/6 rthanol, dried, sprayed wit.h wut,er at the bottom I.5 cm of the plate, chromatographed part of the way w&h I >I-pyridinium formate, then to the top of the plate with 2 t)o 2.2 M-pyridinium form&r. (‘ellulose (MN300) and PEI-cellulose (MN2100) were purchased from Brinkman Instruments. Subsequent nuclease digestion and analysis of oligonucleotides, in additiotl t,c) minor hase analysis, was by the method of Barrel1 (1971). Mononucleot,ides from T, KNase digest,ion bve+rt! sometimes analyzed by chromatography on PEl-cellulosc~ t hill-layer chrornatograpllJ, plates (plastic-backed, purchased from Brinkmall Instruments) rising 0.3 AI-lithium formate (pH 3) as the developing buffer (K. Danna, personal comlnrmicat,it,rl). This lattrr method was very useful for identifying the dinucleotidchs (‘mTT 21nd m7GU.

(e) Transfer RNA analysis

by 12PJc-5 c:h.rornatuyru$q

Mixed tRNA was charged with either 14C or 3H-labeled amitlo acids (New JJt~glantl Nuclear) according t’o Yarus 85 Mertcs ( 1973). U nfractionat.ed aminoacyl-tRNA sytlthetases were prepared a,ccording to tllo rnethotl of Mllelrclr & Borg (1966) througll thc~ DEAE-cellulose chromatography. Charging react,ions \vr:rf’ tcarrtlinatrtl by t,xtractioll witI, water-saturated phenol; the tRNA in t,he aqueous layer LVRStllwl precipitat,ed t,wice at - 30°C wibh I. iw-Na(>l alld 2 x-01. 95% ethanol. RPC-5, the column chromatographic packing used fi)~ thci fractiutlat,iorl of tKNA (Pearson et al., 1971), was purchased from Miles Biochemica81s.

3. Results (a) Isolation

of [ 32P]tRNrlsTrD

Cultures (at about 2 to 3 x 108 cells/ml) ofLS1074. LS1075 and LS1076 were allowed to incorporate 60 to 75:/A of the 32P0, (3 h at 37°C). RlVA was immediately ext,racted and precipitated with ethanol; then each precipitate was dissolved in polyacrylamide gel sample buffer, divided into two approximately equal samples, and each sample was loaded onto a 10% polyacrylamide gel. Previous experiments have shown that tRNATrp migrates quite rapidly in this first-dimension gel (lkemura & Nomura, 1977; G. Barth & S. P. Eisenberg, unpublished results); thus. it, was only necesssry to run the second-dimension in a, small section f’rom thv tmtJtonr of’ the lO’$, gel. An ~xutoradiogram of the second-dimension gel is shown in Figurcb 2’. Sinorb some structural differences were known to exist betwien tRNA TrP from LS1074. Ml075 and I,81076 (Yanofsky

& Soll,

1977;

Hirsh,

1971),

we expected

that

tjht: three

t,RNATr*

variants

might have different electrophoret,ic mobilities on polyacrylamidr gels. However. t)he strains arc in the same spots corresponding to tRI\;A Trp from each of t,he different relative position on the gel, indicating that, any possible differences in structure are not the basis for separation in tht> two-tlilrlellsioila,l gel system, Each sample of tRNATrP was eluted from the gel with about 900;, rrcove~~~. The tckal radioactivity found in tRNATrp from LS1074 (@IX + , su-) was 12% x IO5 ct,s/min (0.3Sgb of total incorporated radioactivity), from LS1075 (trpX--. su - ). 1 1.3 x 105 cts/min (0.35%) and from LS1076 (trpX-, su&,), 9.3 x 10” cts/min (0.35yb). The yields of tRNATrp normalized to total 32P0, incorporated into trichloroacetic acid-insoluble material (in parenthesis) indicates that the level of tRl?A TrP in each of the three strains is the same.

El

T Jti L-

Cellulose

acetate,

pH 3.7 -

acetate,

pH 3.7

e

(b)

t sl 9= = 8 & d

L

Cellulose

acetate,

pH 3.7 b

I!IG. 3. ‘I?, RNaso fingerprints of: (a) LS1074 (wild-typo) t.l~NAT“n; (b) LS1075 (trpY--) tslLNA”r”; (b) and (c), spot a is due to an impurity which (o) LS1076 (trpX-, su&) tRNA Tag. In fingerprints appears only rarely. In tigerprint (b) spot b is a smell fraction of the nucleotide U-omU-C-C-A-A;1.-A-C-C-G which had remained at the origin. The marker dyes B and P are drscribed bq’ Barrel1

trpX

MUTATION

AFFECTS

tRNA

MODIPICATIOS

116

FIG. 2. An autoradiograph showing:the fractionation of selected L3’l’]tRNAs by d-dimenknal gel electrophoresis. Each sample (see text) was electrophoresed t)hrough a 10% polyacrylamide gel (1st D). A small section of each lane containing only the fa,st moving bands was cut out of the gel, turned SO”,and re-electrophoresed in a 2nd dimensional gel (2nd I)) (see Materials and Met,hods) . WT, LS1074 tRNAs; TRP ,Y, LS1075 tRNAs; X, Su+ ITGA, LH1076 tRNAs. Purified [:‘?ItRNATrp is t,he lowest spot on the 2nd dimension gel.

(b) Sequence analysis

of tRNAsTip

T, RNase fingerprints of tRNA Trp from each strain are shown in Figure 3. tRNAT*P from LS1074 gives the fingerprint of wild-type tRNATpp. Also, each spot gives the expected products when redigested with pancreatic RNase and electrophoresed on DEAE paper at pH 3.5 (Hirsh, 1971). fingerprint. The fingerprint of LS1075 tRNA Trp is verv., similar to the wild-type The only consistently observed difference is in the position of spot I which is higher and to the left of its position in the wild-type fingerprint. Redigestion with pancreatic RNaso and subsequent analysis reveals no difference in the products from the spot

trpX

MUTATION

AFFECTS

tRNA

MODIFICATION

117

with altered and normal mobility on the fingerprint and therefore suggests an undetectable or labile modification difference between LS1074 and LS1075 tRNATrp. Digestion of LS1076 tRNATrP yields a T, fingerprint having two spots which are different from those of the wild-type. One spot (spot II) is the oligonucleotide A-A-C-A-C-C-G, which replaces the oligonucleotide C-A-C-C-G in the wild-type molecule. This is due to the previously demonstrated G to A base-change in the D stem of the UGA suppressor tRNA TrP (Hirsh, 1971). The other difference is spot I, which is in a position similar to spot I from LS1075 tRNATrp; thus, the trpX mutation is active in the UGA suppressor-containing strain (spot I also gives the expected pancreatic RNase products). Pancreatic RNase fingerprints of tRNA Trp from each strain are shown in Figure 4. Comparison reveals that the oligonucleotide A-A-A-A-C has moved in the two trpX (LS1075 and LS1076) fingerprints when each is compared to the wild-type pattern. The sequence A-A-A-A-C is in the spot I oligonucleotide from the T, fingerprints, so the change in position of spot I is apparently due to a structural difference in the sequence A-A-A-A-C. The A-A-A-A-C oligonucleotides from both T, (via secondary digestion with pancreatic RNase) and pancreatic RNase fingerprints were redigested to single nucleotides with RNase T, and analyzed by electrophoresis at pH 3.5 on DEAE paper. Aut.oradiography (Fig. 5) shows that the wild-type tRNATrp contained a C, several As. and a smeared spot with an R, (mobility relative to U) of 0.5, a mobility consistent with the presence of 2-methylthio-N6-(A2-isopentenyl) adenosine monophosphate (ms2i6A) (Barrell, 1971). The A-A-A-A-C oligonucleotide from the two trpX strains has a C, a few As (but the A : C ratio is greater here than in the wild-type sample), but lacks a spot at R, = 0.5. If N6-(A2-isopentenyl)adenosine monophosphate (i6A) were present, a spot with an R, of 0.9 (Barre& 1971) would be visible, and if a base modified with a sulfur-containing group were present, a smeared spot should be seen (Hirsh, 1971). We conclude that the A-A-A-A-C oligonucleotide from LS1075 and LS1076 tRNAT’p is unmodified, in contrast with LS1074 (wild-type) tRNATrP, where one of the As in the A-A-A-A-C sequence is the hypermodified base, ms2i6A. According to Hirsh (1971), the second A in this sequence is the normally modified one. Figure 6 shows tRh’ATPp in the cloverleaf pattern and indicates the change due to the trpX a UGA mutation at position 37, and the base-change which makes the tRNA suppressor. (c) RPC-5 column chromatography

of aminoacyl-tRNAs

Since the hypermodified base ms2i6A is found in all E. coli tRNAs which respond to codons beginning with a U (Nishimura et al., 1969 ; Gauss & Sprinzl, 1978), it seemed possible that all those tRNAs (not just tRNA*‘P) would be effected by the trpX mutation. To test this idea, several samples of tRNA extracted from trpX (LS1075) and wild-type cells were charged with various amino acids labeled with 3H or 14C, then co-chromatographed on an RPC-5 column. The result of this experiment is

(1971). Hyphens omitted from Figs for clarity. P.E.1..cellulose: (a) 3 cm in 1 M-pyridinium for-mate, 7 ~-urea, remainder in 2.2 M-forma& and 7 ~-urea; (b) 4 om in 1 M-form&e, 7 M urea, remainder in 2 M-form&e, 7 M-urea; (c) 4 cm in 1 M-formate, 7 M-urea, remainder in 2 M-formate,

7 M-Urea.

FIG. 4. Pancreatic

RNase

fingerprints.

(a), (b) and

(c) are as in Fig.

3. Marker

dyes

B, P and Y are described

by Barrel1

(1971).

trpX

MUTATION

AFFECTS

tRNA

MODIFICATION

119

FIQ. 5. Analysis of the oligonucleotido A-A-A-A-(’ in tRNA TrlJ from LS1074 (lane W), LS1075 (lane X) and LS1076 (lane -XUGA). .a, (1 antI 11 imliratr t hv posit ions of marker mdeotides (Rarrell, 1971). 0 is the origin.

shown in Figure 7. Tryptophanyl(Trp), phenylalanyl(Phe), and tyrosyl(Tyr) tRNAs are all affected by the tr@Z mutation, the others, namely isoleucyl(Ile), methionyl(Met), and valyl(Va1) tRNAs were not altered. Similar results were obtained when tRNAs from LS1273 (tr@-) were compared to those from LX1074 (t&Y+), i.e. Trp-tRNA and Phe-tRNA from the trpX- strain eluted from RPC-5 at lower ionic strength than the isoaccepting species from trpX+ bacteria while Val-tRNA was unaffected by the trpX- allele (data not shown). In addition, [l*C]Trp-tRNA from LS1273 and [3H]Trp-tRNA from LS1075 were compared directly by co-chromatography on RPC-5. The acid-precipitable 14C13H

120

S. P. EISENBERG.

M. YARUS

I

AND

I,.

SOLL

I

U* A CW ‘! And? iJ ’ C.@

(- ’

A)

Fro. 6. The nucleotide sequence of tRNAT”‘. Wild-type tRNA TrR iu shown with changes due to the trpX (--f, A) and UGA suppressor (- “GA+ A) mutations indicated.

ratio remained constant across the single peak of radioactivity, indicating that the tRNAT’p from these two trpX- strains is missing the same modification. Since LS1273 and LS1074 are isogenic except for the trpX region we conclude that this locus is responsible for the change in chromatographic behavior. A comparison of try, operon enzyme levels between these two strains showed the difference expected from reduced attenuation in the trpXmutant background (G. Zurawski & C. Yanofsky, personal communication). Thus both the observed alteration in tRNA structure and the affect on trp operon regulation can be attributed to the trpX mutation. ms2i6A is the only modified base normally found in the three tRNAs affected by trpX- (i.e. tryptophan, phenylalanine and tyrosine) and not found in the three tRNAs unaffected (i.e. isoleucine, methionine and valine) (Gauss & Sprinzl, 1978) by the trpX mutation. For example, Trp-, Phe- and Tyr-RNAs normally all have a 4thiouridine at position 8 (s4U,) so the trpX mutation could conceiveably lead to a loss of that modification in addition to the ms2i6A. However. both Val-and Met-tRNAs have an s4U, so they too should be affected by the trpX mutation; but they are not. In this way, all modifications which Trp-, Phe- and Tyr-tRNAs have in common, except ms2isA, can be eliminated as a cause of the shift in elution on RPC-5. These results are consistent with the proposal that the trpX gene codes either for a tRNAmodifying enzyme or for an enzyme required for the synthesis of a substrate of this modification system. Sequences presented above indicate that the UGA suppressor tRNATrp from LS1076 (su+trpX-) lacks hypermodification. Thus, the trpX mutation in this strain should affect the elution of tRNAs from RPC-5 in the same way as it does in LS1075 or IL51273 (su-trpX-). Figure 8(a) and (b) shows that Trp- and Phe-tRNAs from

trpX

MUTATION

AFFECTS

tRNA

IO 000

121

0

Vol IGUX)

5000 0

MODIFICATION

t

@-8-e-e-e-8-8--8

P

‘\

@-*8-8-8--8-8--Q-8

Met (AUG)

2000

Phe (UUUK)

0 400 -

Fraction

no.

FIG. 7. Comparison of several tRNAs from LS1074 (au-, trpX+) and LS1075 (SW, trpX-) by RPC-5 chromatography. Samples of LS1074 tRNA were charged with 14C labeled amino acids as indicated in the Figure. LS1075 tRNA was amino-acylated with 3H-labeled amino acids. A mixture of LS1074 and LS1075 tRNAs (charged with the same amino acid) was dissolved in 0.5 ml of 1.4 mM-/%mercaptoethanol) +0.3 RPC-5 buffer (50 mmsodium acetate, pH 4.5, 10 mM-MgCl,, M-NaCl, applied to a 0.9 cm x 56 cm RPC-5 column at 20°C, t,hen eluted with a 300-ml linear gradient from O-3 M to 1.3 ix-NaCl (in RPC-5 buffer). Ten-ml fractions were collected, precipitated wit,h 2 M-HCl, filtered and counted by scintillation spectrometry. Determination of radioactivity in each fract,ion included correcting for spillover of cts/min from one isotope into the “window” of the other isotope. Codons are in parentheses. --o-o--, 3H; -e-a-, 14C.

LS1076 are chromatographically affected by trpX-, and in Figure 8(c), where Phet,RNAs from LS1075 and LS1076 are compared directly, they co-elute from RPC-5. The results reported here are consistent with the recent work of B. S. Vold (personal communication). She found that total RNA from trpX strains contains less than 10% of the wild-type level of isopentenyl nucleosides. This determination was performed by binding to an antibody specific for the isopentenyl modification. In addition, no detectable sulfur was found associated with the normally hypermodified base when cells conisolated from trpX cells grown in 35S-labeled medium, whereas wild-type tained normal sulfur levels.

122

H. P. EISENBERG, I

M. YARUS I

AND I

(a)

I,. SOLI, I 300

2ooc

200

I ooc I00

0

0

0

T E ICQO ;‘, E * nz 7 ; 500 g .c E 0 ‘37 e 0

12 ooc YE E \ v) 2 : +i 5. :: .-E E a

8000

4000

F LEJ

600

900

600

300

3

Fraction

no.

FIG. 8. Comparison of L81076 (su&,, ti-pX-) t~RNA to LS1074 (SW, trpX+) and LS1075 (su, trpX-) tRNA by RPC-5 chromatography. The same procedure was followed as in Fig. 7 except : in (a) and (b), LS1076 tRNA is charged w&h 3H-labeled amino acids; in (c) LS1075 tDNA was charged with [14C]phenylalanine whik Ml076 t)DKA was charged with [3H]phen,ylalaninc, the c\-- -C NaCl gradient was from 0.55 M to 0.95 M, and the volume of each fract,ion wax 5 ml. . 3H;

-e-o-,

14C.

4. Discussion The function of the hypermodified base, ms2isA, has been studied by several groups. Gefter & Russell (1969) showed that a lack or even a partial lack of modifications on the normally hypermodified base reduced the efficiency of tRNATYr binding to ribosomes in the presence of the appropriate trinucleotide codon. Recently, Grosjean et al. (1976) have provided a possible physical explanation for t’his effect. They studied the binding interaction between tRNAs with complementary anticodons, and found that tRNAPhe

tTpx

MUTATION

AFFECTS

tRNA

MODIFICATION

12.3

(anticodon GAA) from yeast or E. co& each having a hypermodified base next to the anticodon, bind much more strongly to t’RNAGIU (anticodon UUC) than does tRNAPhe from Mycoplasma, which does not have a hypermodified base. Finally, Laten et al. (1978) found that an antisuppressor mutant of Sacchmomyces cerevisiae contains tRNA which completely lacks the modification of the normally hypermodified base i6A. Since the affected suppressor tRNA normally has an i6A, the loss of that hypermodification apparently causes a reduced efficiency of suppression, presumably during ribosomal interaction. We can now explain the trpX effect on attenuation. As described above, tRNATPP isolated from trpX bacteria has an unmodified A at’ the 3’ side of the anticodon base ms2i6A. Since this (position 37), where normal tRNA Trp has the hypermodified modification has been strongly implicated as a requirement for efficient tRNAribosome interactions, its absence, due to the trpX mutation, may produce inefficient translation at the tandem tryptophan codons in the leader sequence RNA. This is consistent with the translation model of Lee & Yanofsky (1977). since an inability to read those trp codons efficiently should lead to increased expression of the trp operon. The UGA suppressor tRNA Trp must have an unusual ability to interact with the ribosome in response to codons of the group UGX (Buckingham & Kurland, 1977). since the tRNA can efficiently read the codons UGG, UGA and UGU, one of the cysteine codons. The suppressor tRNA must &ill retain. at least partially, its unusual ribosomal translation property while lacking (hgper) moditications, since this tRNA in a trpXstrain (i.e. LS1076) can suppress a UGS codon (Yanofsky & Soll, 1977). We suggest that, because of its unusual property. the UGA suppressor tRNATrp can efficiently translate the tandem UGG codons despite the lack of hypermodifications. The analysis of the trpX mutation presented here direct’lp relates attenuation to translational efficiency, due to the well-documented funct’ion for the hypermodified base, ms2i6A, and the previously known effect of the UG-4 suppressor mutation on the ribosomal interaction. Studies on the regulation of several biosynthetic enzymes in bacterial strains containing the hisT mutation can also be discussed in this context. First isolated and characterized in i3almoneEla typhimurium (Chang et al.. 1971; Singer et al., 1972) and lat#er in E. coli by Bruni et al. (1977), strains carrying the hisT mutation produce tRNAs lacking the modified base, pseudouridine (Y) in t,he anticodon loop. Many tRNAs normally have this modification: and it has been demonstrated that deregulation of the synthesis of biosynthetic enzymes specific for amino acids which are cognate to several of these tRNAs occurs in hisT mutants (Brenchley & Williams, 1975). Recently, the h&T mutation has been shown to induce reduced efficiency of suppression by those suppressor tRNAs which normally have the relevant !!% (L. Bossi X: J. R. Roth, personal communication). Thus: the hisT-dependent deregulation of several operons could be analogous to the effect of trpX on t~he trp operon, especially with regard to deregulation of the his operon, since the his leader sequence has seven consecutive histidine codons (Di Nocera et al., 1978 : Barnes, 1978) and tRNAHiS is one of those tRNAs which lacks Ys (in the anticodon loop) due to the h&T mutation (Singer et al., 1972). It has been pointed out that yeast strains which lack i6A (in tRNA) grow as well as non-mutants under a variety of conditions (Laten et al., 1978). We have compared the growth rate of the trpX strain to its isogenic wild-type strain and found little difference in doubling times or rate of uptake of 32P04 into nucleic acid (see Results). C. Yanofsky

124

S. P. EISENBERG,

M. YARUS

,4ND

L.

SOLL

(personal communication) has found that trpX mutants grow only slightly (10 to 200/‘) slower than wild-type strains. Since mutations which lead to the lack of hypermodified bases on tRNA have little effect on overall growth, protein synthesis in general is probably only slightly affected. This is at least partially due to the fact that only six species of tRNA contain the i6 modification. Translation of the trp leader is presumably affected to a larger degree since the presence of two UGG codons magnifies the decreased efficiency of translation by tRNA TrP lacking hypermodifications. Alternatively. hvpermodifications could br important for translation of codons in a particular contkxt. Thus, tRNATr* might, need its hypermodified base only to efficiently t.ranslate a trp codon which is adjacent. to another trp codon. Tandem tryptophan codons must, be extremely rare (only loi;, of the amino acids in total E. coli protein is tryptophan) so the lack of hypermodifications would have little effect on total protein synthesis. but in the trp operon leader. where tandem UGGs exist, the effect on translation and subsequent, regulation, is observed. There is the possibility that these tRNA modifications function pey se as regulatory effecters, because undermodified tRNA is synthesized when protein synthesis is restrained. In particular, ms2i6A is not made during leucine starvation (Kitchingmall & Fournier, 1977), during chloramphenicol treatment (Huang $ Mann, 1974). in phage-infected cells after ultraviolet irradiation (Gefter & Russell, 1969 ; D. Bradley. personal communication), and we have observed partial undermodification during slow growth in a minimal medium. Also, when cultures of 8. typhimuriuwl. were exposed to an amino acid shiftdown (or after other changes in growth medium which produced nutritional stress), the tRNA Phe from these bacteria was eluted from RPC-5 at lower ionic strength (as if it lacked hypermodification) (Turnbough et al., 1979). These observations could mean that hypermodification of nucleotides in the anticodon loop is more sensitive to partial inhibition of protein synthesis than is the synthesis of tRNA. Perhaps the modification pathway includes an unstable element which decays during such stress. There are indications that the tryptophan (see above)? phenylalanine (Zurawski et al., 1978), threonine (Gardner, 1979) and histidine (Di Niocera et al.. 1978; Barnes. 1978) biosynthetic operons. and perhaps others (Brenchley Kr Williams, 1978), use similar attenuation controls. The result would therefore be to increase synthesis ot some amino acids whenever RNA synthesis proceeds during restraint of protein synthesis by, e.g., amino acid starvation. This proposed regulatory response would be slow, and depends on synthesis of tRNA. but might be adaptive. Others have shown that the hypermodified base is isopentenylated prior to the attachment of the sulfur-containing groups (Gefter & Russell, 1969 ; Agris et al.: 1975). Alternatively, Vold (1979) found a low level of ms2A in tRNA-derived nucleosides from a trpX strain, indicating that in this mutant the normal sequential process needed to produce ms2i6A is not strictly adhered to. In light of this and the results reported in this paper, we propose that the designation trpX be changed to m.iaA to indicate the first known gene in E. toll: required for the synthesis of 2-methylthio-6isopentenyl adenosine. The authors wish to thank Drs Charles Yanofsky and Gerard Zurawski for providing the bacterial strains used in this work and for evaluating and comme&ng on the manuscript prior t,o publication. We also thank Doug Bradley and George (Chip) Barth for

practical

help during the early phases of this study.

trpX

MUTATION

AFFECTS

tRNA

MOUIPICATION

125

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