Proc. Natl. Acad. Sci. USAVol. 90, pp. 2305-2309, March 1993Biochemistry

From elongator tRNA to initiator tRNA(tRNA identity switch/methionine tRNA/glutamine tRNA/formylation/ribosomal P site)

UMESH VARSHNEY, CHAN PING LEE, AND UTTAM L. RAJBHANDARYDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by H. Gobind Khorana, December 10, 1992

ABSTRACT We show that the two most important prop-erties needed for a tRNA to function in initiation in Escherihiacoli are its ability to be formylated and its ability to bind to theribosomal P site. This conclusion is based on conversion of twodifferent elongator tRNAs to ones that can act as initiators inE. coli. We transplanted the features unique to E. coli andeubacterial initiator tRNAs to E. coli elongator methioninetRNA (tRNAMet) along with an anticodon sequence change andanalyzed their activities in initiation in E. coli. Introduction ofa CloA72 mismatch at the end of the acceptor stem oftRNAMet,which generates the minimal features necessary for formyla-tion, produces a tRNA with very low activity in initiation.Subsequent introduction of three consecutive G-C base pairs atthe bottom of the anticodon stem, which is necessary forribosomal P site binding, produces a tRNA with significantactivity in initiation. Furthermore, introduction of the featuresnecessary for formylation and for ribosomal P site binding intoE. coli elongator glutamine tRNA produces a tRNA thatinitiates protein synthesis in E. coli.

Of the two classes of methionine tRNAs present in allorganisms, the initiator is used for initiation of proteinsynthesis, whereas the elongator is used for inserting methi-onine into internal peptide linkages (1). Because of theirunique function, initiator tRNAs possess several propertiesdistinct from those of elongator tRNAs. In eubacteria and ineukaryotic organelles such as mitochondria and chloroplasts,the initiator is used as formylmethionyl-tRNA (fMet-tRNA).Following aminoacylation of the initiator tRNA (tRNAfMet),the methionyl-tRNA (Met-tRNA) is specifically formylatedto fMet-tRNA by methionyl-tRNA transformylase. ThefMet-tRNA binds to the P site on the ribosome in contrast toelongator tRNAs, which first bind to the A site (2, 3). Inaddition, initiator tRNAs do not bind to the A site on theribosome and therefore cannot act at the elongation step ofprotein synthesis.Along with these special properties, eubacterial initiator

tRNAs also possess unique sequence and/or structural fea-tures that are not found in elongator tRNAs (4). Theseinclude: (i) the absence of a Watson-Crick base pair betweennucleotides 1 and 72 at the end of the acceptor stem, (ii) thepresence of a sequence of three guanines and three cytosinesat the bottom of the anticodon stem forming three consecu-tive G-C base pairs, and (iii) the presence of a purine-11pyrimidine-24 base pair in the dihydrouridine stem incontrast to a pyrimidine-11purine-24 base pair in othertRNAs.

Functional studies on mutants of Escherichia coli initiatortRNA have provided information on features important forspecifying their distinctive properties (5, 6), including thesequence and/or structural features that are crucial forformylation and for binding of the tRNA to the P site on theribosome. The key features necessary for formylation appear

to be a mismatch or a weak base pair between nucleotides 1and 72, a G2'C71 base pair, and'a C3'G70 base pair (7, 8).Mutations in G4'C69 and A11U24 base pairs affect formyl-ation kinetics but have less of an effect than mutations at theabove positions (7). Once the tRNA is formylated, the featureimportant for targeting the tRNA to the P site on the ribosomeappears to be the three consecutive G'C base pairs in theanticodon stem (9).Given the many distinctive properties of initiator tRNAs,

a question of interest is: what are the minimal alterations inan elongator tRNA that are needed to convert it into aninitiatortRNA? In this paper we show that introduction ofthefeatures necessary for formylation of the tRNA and fortargeting the tRNA to the P site on the ribosome allows theE. coli elongator methionine tRNA (tRNAMet) and glutaminetRNA (tRNAGin) to initiate protein synthesis in E. coli.

MATERIALS AND METHODSMutagenesis of Elongator Methionine and Glutamine tRNA

Genes. Mutant tRNA genes were generated by using oligo-nucleotide-directed mutagenesis (6, 9). For mutant elongatormethionine tRNA genes, a 93-nucleotide-long mutagenicprimer was used to replace the (C27A28T29C30A31)'(T39G40A41T42G43) mutant initiator tRNA gene present inphage M13 DNA (7) with' the CloA72 mutant of E. colielongator methionine tRNA gene. The resulting mutant elon-gator methionine tRNA gene was then used to generate theother elongator mutant tRNA genes listed in Table 1. Formutant glutamine tRNA gene, a 93-nucleotide-long muta-genic primer was used to replace the T35A36 mutant initiatortRNA gene present in'M13 DNA. The mutant tRNA geneswere characterized by sequencing of the entire tRNA gene.All of the mutant tRNA genes have the same promoter andterminator sequences as in the initiator tRNA gene.

Vectors and Strains Used. The mutant tRNA genes usedwere cloned into either pRSVCATaml.2.5 (a pBR322-basedvector) or pTZ19R (a pUC-based vector). The E. coli genesencoding glutaminyl- and methionyl-tRNA synthetases werecloned into pACl (a pACYC-based vector) (10). The E. colistrains used were either CA274 (9) or TG1.

Assays for Initiation Activity of Mutant Elongator Methio-nine and Glutamine tRNAs. The mutant tRNAs studied hereall have a CAU -* CUA anticodon sequence change. Thisallows assay for their ability to initiate protein synthesis fromUAG by using a mutant chloramphenicol acetyltransferase(CAT) gene (CATaml.2.5) in which the initiator AUG codonis changed to UAG (10, 11). Production ofCAT protein wasmeasured by assay for CAT activity in cell extracts and byimmunoblot analysis (11).

Detection of Mutant tRNAs by Northern Blot Hybridization.Total tRNAs isolated from E. coli at pH 8.0 and at roomtemperature were fractionated by gel electrophoresis on a12% nondenaturing polyacrylamide gel as described (7) priorto hybridization.

Abbreviation: CAT, chloramphenicol acetyltransferase.

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2306 Biochemistry: Varshney et al.

Table 1. List of E. coli elongator methionine tRNA mutants

Mutantdesignation Nature of mutations

M:il CloA72/T35A36M:i2 CloA72/(G29G30G31).(C39C40C41)/T35A36M:i3 CloA72/AllT24/(G29G30G31PC39C40C41)/T35A36

Separation of Uncharged, Aminoacyl-, and Formylami-noacyl tRNAs. Total tRNAs isolated from E. coli under acidicconditions were subjected to polyacrylamide gel electropho-resis at pH 5.0 and at 4°C (12). The various forms of thetRNAs were detected by Northern blot hybridization byusing sequence-specific deoxyribooligonucleotide probes la-beled at the 5' end with 32p.

RESULTS

Mutants of E. coli Elongator Methionine tRNA. Threeelongator tRNA mutants were used (Fig. 1 Left and Table 1).These mutants, designated M:il, M:i2, and M:i3, containedrespectively one, two, or all three of the features unique to E.coli initiator tRNA. M:il contained a CloA72 mismatchinstead of a G1-C72 base pair. Because the second and thirdbase pairs in the acceptor stem of E. coli tRNAMet andtRNAfmet are identical, the M:il mutation generates theminimal features necessary for recognition of the tRNA bythe formylating enzyme. M:i2 contained additional changesin the anticodon stem such that it has three G-C base pairs atthe bottom of the anticodon stem. M:i3 further contained anA11U24 base pair in the dihydrouridine stem instead of aC11G24 base pair.

For functional studies in initiation from UAG codon invivo, the above mutant tRNAs also contained an anticodonsequence mutation from CAU -- CUA (11). Because of the

anticodon sequence change, all three of the elongator tRNAmutants studied are very poor substrates for E. coli methio-nyl-tRNA synthetase (6, 13, 14). Although the elongatortRNA mutant with the anticodon change alone is aminoacy-lated in E. coli with lysine (15), the effect on aminoacylationwith lysine of the CloA72 mismatch common to the threemutants studied here is'unknown (16). Since the E. coliinitiator tRNA mutant with the same anticodon sequencechange is aminoacylated with glutamine in vitro and in vivo(6, 12, 13) and the presence of a CloA72 mismatch favorsaminoacylation by E. coliglutaminyl-tRNA synthetase (6, 17,

AOH

c

AC -pG-C -A

G-C

C-GU-A

A-U

C-GA G-C UA

GA t As UGCCCA

D CUCG G

Gm ACAGGT,CG c~x7

GDDAGAGC A CGGGmTGG U - A -**CG- C -G C

G4- A-jr PCC A

U t6A

04C A U

i A36

T35

t RNA Mmet

AOH

cCG ---- A

C *-pU A

G-C

C4-G-C -_GG-CG-C

U-A

A-U

S GCUCcU A

cG ACCG 11l GGm CGAGGT C

GDAAGGCA C-GC AUC-G

G-CG-C

G A CC -Um --A

U m2A

C U G>,A36

tRNAGln

FIG. 1. (Left) Sequence of E. coli tRNAMeC in cloverleaf formwith the sites of mutation indicated by arrows. The mutants de-scribed here have in common the T35A36 mutation in the anticodon

sequence and the C1oA72 mutation in the acceptor stem (Table 1).(Right) Sequence of E. coli tRNA2'n in cloverleaf form with the sites

of mutation to generate Q:i2 indicated by arrows.

18), it is likely that these mutant tRNAs are aminoacylated atleast to some extent with glutamine. Also, we had found thatsome mutants of E. coli initiator tRNA that are defective informylation and inactive in initiation when aminoacylatedwith glutamine become partially active in cells overproducingmethionyl-tRNA synthetase (19). Therefore, we studied theeffect of overproduction of either enzyme on activity of themutant elongator tRNAs in initiation in E. coli.

Activity of the Mutant Elongator Methionine tRNAs inInitiation in E. col. Extracts from cells containing theCATaml.2.5 reporter gene and one of the mutant tRNAgenes were assayed for CAT activity and CAT protein levelsas a measure of the activity of the mutant tRNAs in initiation.Table 2 shows that none of the three elongator tRNA mutantsare active in initiation in cells that are not overproducing anyof the aminoacyl-tRNA synthetases (column -) or in cellsthat are overproducing glutaminyl-tRNA synthetase (+GlnRS). In cells overproducing methionyl-tRNA synthetase(+ MetRS), however, the M:il mutant with the acceptor stemchange has a low but detectable level of activity. In contrast,the M:i2 mutant with additional changes in the anticodonstem is quite active. The CAT activity in cell extracts is -30%of that in cells transformed with a mutant initiator tRNAcarrying the same anticodon sequence change. Interestingly,when the third unique feature of the initiator tRNA, anAl1U24 base pair, is introduced to generate the M:i3 mutant,no CAT activity is found in cell extracts. The reason for thisis that these cells do not accumulate any M:i3 mutant tRNA.Northern blot analysis (Fig. 2) of tRNA isolated from trans-formants carrying the M:i2 or the M:i3 mutant tRNA genesshow that while the M:i2 mutant tRNA is overproduced invivo, there is no accumulation of M:i3 tRNA (compare lanes2 to 1 and 4 to 3). Sequence analysis and in vitro transcriptionof the M:i3 tRNA gene indicate that the promoter for thetRNA gene is intact and is active (data not shown). Resultsof in vitro processing experiments on the M:i3 precursortRNA suggest that the precursor and/or the mature tRNAmay be unstable in E. coli extracts (data not shown). It ispossible that the AllU24 base pair can exist only in certainsequence context(s) and that these are not present in E. colitRNAMet.Immunoblot analysis (20) of cell extracts for CAT protein

(11) confirms the assays for CAT activity (Fig. 3). CATprotein is found in transformants that carry either the E. coliinitiator tRNA mutant (fM/T35A36) or the elongator tRNAmutant M:i2. Synthesis of CAT protein with the M:i2 mutantrequires overproduction of methionyl-tRNA synthetaseTable 2. Relative CAT activities in extracts of transformedE. coli

Relative CAT activity, %tRNA gene - + GlnRS + MetRS

fM/T35A36* 100 118 263M:ilt nd nd 3M:i2 0.8 0.8 81M:i3 0.3 0.1 0.2Q:i2 10 15 35

Relative CAT activities (%) in extracts of E. coli CA274 trans-formed with pRSV plasmids carrying the CATaml.2.5 gene andvarious mutant tRNA genes including those listed in Table 1. Thetransformants also contained another plasmid that did not (-) or did(+) contain the gene for E. coli glutaminyl-tRNA synthetase (GlnRS)or E. coli methionyl-tRNA synthetase (MetRS).*E. coli initiator tRNAfmet gene carrying the T35A36 mutation. CATactivity in cells containing this tRNA gene and not overproducingany of the aminoacyl-tRNA synthetases is fixed at 10.

tnd, Not determined. However, since these cells do not grow on

plates containing chloramphenicol after 16 hr, they contain muchless than 4% CAT (11).

Proc. Natl. Acad Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 2307

1 2 3 4itRNA Gene fM/

T35A361 M: 12 M: j3 M l

LMetRS ± 1 I L

.S.......U.......

Ip*F,

/ - loctomase -

CAT --H

FIG. 2. RNA blot hybridization of total tRNA isolated fromtransformants carrying the M:i2 (lanes 1 and 3) or the M:i3 (lanes 2and 4) mutant elongator methionine tRNA genes on a pBR-basedvector (lanes 1 and 2) or a high-copy pUC-based vector (lanes 3 and4). The probe used was complementary to nucleotides 25-45 of theM:i2 or the M:i3 mutant tRNA.

(compare third and fourth lanes from left). In accordancewith the CAT activity assays, a very small amount of CATprotein is present in extracts of cells that carry the M:ilmutant and are also overproducing methionyl-tRNA synthe-tase (Fig. 3, rightmost lane). The similar levels of,-lactamasein the cell extracts suggest that the transformants all con-tained the vector carrying the CATaml.2.5 gene and therespective mutant tRNA genes (11).

Aminoacylation and Formylation ofM:i2 Mutant tRNA inE.coli. Recently, we described a method (12) to separateuncharged, aminoacylated and formylaminoacylated formsof tRNA from each other and to detect these various formsby using Northern blot analysis of tRNA isolated underconditions where the linkage between the amino acid and thetRNA is preserved. Application of this method to the M:i2mutant tRNA shows that -50% of the M:i2 mutant tRNA isaminoacylated in vivo but none of it is formylated (Fig. 4, lane3). In cells overproducing glutaminyl-tRNA synthetase,somewhat more of the tRNA is aminoacylated, presumablyat least partially with glutamine; however, there is still noformylation of the aminoacyl-tRNA (lane 4). In contrast, incells overproducing methionyl-tRNA synthetase, a substan-tial fraction of the tRNA is now converted to the formylatedform (lane 5). This is most likely because overproduction ofmethionyl-tRNA synthetase leads to aminoacylation of theM:i2 mutant tRNA with methionine in vivo (19, 21-23) andthat this tRNA is a much better substrate for methionyl-tRNAtransformylase than the tRNA aminoacylated with otheramino acids. The formylation of M:i2 tRNA only in cellsoverproducing methionyl-tRNA synthetase would explain itsactivity in initiation in these cells.An alternate possibility is that the E. coli elongator methi-

onine tRNA mutant M:i2 is active in initiation in cells that arenot overproducing any aminoacyl-tRNA synthetase but thatthe CAT protein that is made is unstable because it is initiatedwith lysine. This possibility can be ruled out. Proteins withlysine or arginine at the N terminus are thought to be unstable

FIG. 3. Immunoblot analysis with rabbit anti-CAT and anti-,3lactamase antibodies of crude extracts from E. coli CA274 trans-formants carrying the CATaml.2.5 gene and either the T35A36mutant initiator tRNA gene or the various mutant elongator tRNAgenes as indicated. The transformants also contained another plas-mid which did (+) or did not carry the gene for E. coli methionyl-tRNA synthetase.

in E. coli (24) because they are substrates for the E. colileucine/phenylalanine-tRNA protein transferase (L/F-transferase). This enzyme attaches leucine or phenylalanineto the N terminus of such proteins and thereby destabilizesthe proteins in vivo. We transformed an E. coli strain thatcarries a disruption in the gene for the L/F-transferaseenzyme with the plasmid carrying the pRSVCATaml.2.5gene and the M:i2 mutant tRNA gene. Transformants still do

- GIn-tRNA(?)- Met-tRNA- fMet-tRNA

.. :~~~ tRNA.~~~~~. t.N

FIG. 4. RNA blot hybridization of total tRNA isolated from E.coli transformants carrying the M:i2 mutant elongator tRNA gene.The tRNAs, isolated under acidic conditions, were separated byelectrophoresis on a 6.5% acid-urea polyacrylamide gel at 4°C (12).The probe used was complementary to nucleotides 21-48 of the M:i2mutant tRNA. Lanes: 1 and 2, marker tRNAs isolated from TG1 cellsby using a high-copy pUC vector (lane 1) and treated at alkaline pHto deacylate the charged tRNA prior to electrophoresis (lane 2); 3-5,tRNA isolated from CA274 transformants containing an additionalplasmid that had no aminoacyl-tRNA synthetase gene (lane 3), the E.coli glutaminyl-tRNA synthetase gene (lane 4), or the E. coli me-thionyl-tRNA synthetase gene (lane 5).

Biochemistry: Varshney et al.

2308 Biochemistry: Varshney et al.

1 2 3

aa-tRNA -

faa-tRNA -

tRNA-

FIG. 5. RNA blot hybridization of total RNA isolated from E. coliCA274 transformants carrying the E. coli glutamine tRNA mutant,Q:i2. The tRNAs, isolated under acidic conditions, were separatedby electrophoresis on a 6.5% polyacrylamide gel at 4°C. The probeused was complementary to nucleotides 21-48 of the Q:i2 mutanttRNA. The transformants used contained another plasmid that hadno aminoacyl-tRNA synthetase gene (lane 1), the E. coli glutaminyl-tRNA transferase gene (lane 2) or the E. coli methionyl-tRNAsynthetase gene (lane 3). Indicated on the left are positions ofmigration of tRNA, formylaminoacyl (faa)-tRNA, and aminoacyl(aa)-tRNAs.

not produce any CAT protein as indicated by lack of growthon plates containing chloramphenicol, even after prolongedincubation (48 hr).Mutant of E. coli Elongator Glutamine tRNA. Fig. 1 Right

shows the sites of mutation introduced into the E. coliglutamine tRNA (tRNAGin) to convert it to an initiator tRNA.Besides the anticodon sequence change needed for functionalstudies in initiation in vivo, the mutant contained changesneeded for formylation and for targeting the tRNA to theribosomal P site.The glutamine tRNA mutant Q:i2 is active in initiation in

E. coli (Table 2). In cells overproducing methionyl-tRNAsynthetase, CAT activity in cell extracts is -45% of thatobtained with the corresponding E. coli elongator methioninetRNA mutant M:i2. The higher activity of the Q:i2 mutant ininitiatioh in cells overproducing methionyl-tRNA synthetaseis probably because under these conditions the tRNA ispartly aminoacylated with methionine, and the tRNA carry-ing formylmethionine is a better initiator than the tRNAcarrying formylglutamine (19). Unlike the M:i2 mutant, how-ever, the Q:i2 mutant is not totally dependent on overpro-duction of methionyl-tRNA synthetase for activity in initia-tion (Table 2). Analysis of mutant tRNAs by gel electropho-resis provides an explanation for this result. In contrast to theM:i2 mutant, which is formylated only in cells overproducingmethionyl-tRNA synthetase, the Q:i2 mutant is formylatedunder all conditions tested (Fig. 5, lanes 1-3) with littleaccumulation of aminoacyl-tRNA.

DISCUSSIONWe have shown that introduction of the minimal featuresnecessary for formylation of the tRNA and for directing thetRNA to the P site on the ribosome converts E. coli elongatormethionine and glutamine tRNAs into those that can initiateprotein synthesis in E. coli. Introduction of a CloA72 mis-match into the elongator methionine tRNA, with an antico-don sequence change to allow assay for activity in initiationin vivo, generates the minimal features necessary for form-ylation of the tRNA. This tRNA has a low but detectableactivity in initiation (Table 2 and Fig. 3). Further introduction

of three consecutive G-C base pairs at the bottom of theanticodon stem generates features important for P site bind-ing. This tRNA has significant activity in initiation. Theamount of CAT activity in extracts of cells carrying themutant elongator tRNA is -309o of that in cells carrying theE. coli initiator tRNA mutant with the corresponding anti-codon sequence change.The elongator methionine tRNA mutant M:i2 is active in

initiation only in cells that are overproducing methionyl-tRNA synthetase. The reason for it is that the M:i2 mutanttRNA is formylated only when it is aminoacylated withmethionine (Fig. 4, lane 5). Since the M:i2 mutant tRNA is avery poor substrate for methionyl-tRNA synthetase becauseofthe anticodon sequence change (14), aminoacylation of thistRNA with methionine requires overproduction of the en-zyme. These results are similar to those obtained with theT35A36/G72 mutant ofE. coli initiator tRNA (19). The strongcorrelation between formylation of the M:i2 mutant tRNA(Fig. 4, lane 5) and activity in initiation (Table 2) supports ourprevious conclusion, based on analysis of mutants of E. coliinitiator tRNA, that formylation of initiator tRNA is impor-tant for initiation of protein synthesis (11). Further supportfor this comes from the finding that the Q:i2 mutant derivedfrom glutamine tRNA, which is formylated in E. coli under allconditions (Fig. 5), shows partial activity in initiation, evenin cells not overproducing glutaminyl- or methionyl-tRNAsynthetase. The work of Guillon et al. (25), showing thatdisruption of the gene for methionyl-tRNA transformylase inE. coli results in cells that grow extremely slowly, is alsoconsistent with this conclusion.

In contrast to the methionine elongatortRNA mutant M:i2,which is formylated in vivo only when aminoacylated withmethionine, the glutamine tRNA mutant Q:i2 is formylatedeven when aminoacylated with glutamine (Fig. 5). Thus, theQ:i2 mutant is a better substrate for the formylating enzymethan the M:i2 mutant. Both tRNAs contain the same nucle-otides at positions 1, 72, and base pairs 2-71 and 3 70, whichare considered important for formylation (7, 8, 26). The majordifference is the absence in the M:i2 mutant and the presencein the Q:i2 mutant of base pairs G4+C69 and G5 C68 found inthe E. coli initiator tRNA. Mutations of the G4-C69 andG5 C68 base pairs to C-G base pairs in an initiator tRNAsequence background have a relatively small effect (7),lowering the Vm./Km parameters in formylation by approx-imate factors of 4.7 and 2.2, respectively. However, a similarmutation of the G4-C69 base pair in an elongator methioninetRNA background has a more severe effect, loweringVmax/Km by about 2 orders of magnitude (8). Thus, therelative importance of the G4-C69 and possibly the G5-C68base pairs in formylation depends on the tRNA context (8).The total lack offormylation ofthe M:i2 mutant tRNA in cellsoverproducing GlnRS is, therefore, likely due to the com-bined effect of having glutamine instead of methionine andnot having the G4-C69 and possibly the G5 C68 base pairs inthe tRNA.

The first two authors contributed equally to this work. We thankour colleagues Dr. C. Ming Chow for her comments and suggestionson the manuscript, Dr. Michael R. Dyson for the S100 extracts usedfor the processing reaction, and Annmarie McInnis for carefulpreparation of the manuscript. We thank Dr. Thomas Shrader for theE. coli strain carrying a disruption in the L/F transferase gene. Thiswork was supported by grant GM 17151 from the National Institutesof Health.

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