Vol. 164, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1985, p. 1117-11230021-9193/85/121117-07$02.00/0Copyright © 1985, American Society for Microbiology

Genetic Organization and Transcription from the Gene (trmA)Responsible for Synthesis of tRNA (Uracil-5)-Methyltransferase by

Escherichia coliPAR H. R. LINDSTROM,' DIETRICH STUBER,2 AND GLENN R. BJORK3*

Pharmacia AB, Molecular Biology Division, 5-751 82 Uppsala, Sweden'; F. Hoffmann-La Roche & Co., ZFE, CH4002Basel, Switzerland; and Department of Microbiology, University of Umea, S-901 87 Umea, Sweden3

Received 24 April 1985/Accepted 12 September 1985

The enzyme catalyzing the formation of 5-methyluridine (ribothymidine) in tRNA of Escherichia coli is tRNA(uracil-5)-methyltransferase (EC 2.1.1.35). A 2.8-kilobase EcoRI chromosomal DNA fragment contains tnrA,the structural gene for this enzyme. Subcloning, transcription in vitro, TnS insertion mutagenesis, andtranscriptional fusion experiments were performed to establish the gene organization of the trmA region on theE. coli chromosome. rmA is a monocistronic operon. The trmA promoter was localized by in vitro experiments,and the direction of transcription was shown to be counterclockwise on the standard E. coli K-12 chromosomalmap. The level of transcription of rmA in vitro and the expression of protein in minicells equal those of the blagene of plasmid pBR322.

tRNA like other stable RNAs in the cell, contains modifiednucleosides which are derivatives of the four ordinary nu-cleosides adenosine, guanosine, cytidine, and uridine. Someof the modified nucleosides, like 1-methylguanosine (m'G),are found only in a few tRNA species, whereas others, like5-methyluridine (m5U, ribothymidine), are found in all tRNAspecies of Escherichia coli. The latter modified nucleoside isonly present in the common sequence G53-m5U54-P55-C56,which is part of loop IV. It is present in tRNA from mostorganisms except Archaebacteria spp., which instead havemlP in position 54 of their tRNA. Furthermore some strainsof Mycoplasma completely lack m5U in their tRNA (5). Thebiosynthesis of the modified nucleosides occurs after theprimary transcript has been made. tRNA modification is anintegral part of the biosynthesis of tRNA, which, besidesmodification, includes several endo- and exonucleolyticsteps (11, 21). The enzyme tRNA (m5U)methyltransferase isa tRNA biosynthetic enzyme and uses as substratespolycistronic as well as monocistronic precursor tRNAs (4,27, 28). tRNA from mutants (trmA) which completely lackm5U in their tRNA serves as an excellent and specificsubstrate in vitro (6). As a methyl donor the enzyme from E.coli uses S-adenosyl-L-methionine. We have earlier shownthat the amount of tRNA (m5U)methyltransferase increaseswith increasing growth rate in the same manner as stableRNA. In addition, we have shown that the enzyme isstringently regulated and behaves similarly to stable RNAduring shift-up and shift-down conditions (22, 23). Thus, theregulatory behavior of the tRNA (m5U)methyltransferasehas in all our experiments so far mimicked that of stableRNA. However, in contrast to rRNA and tRNA, the enzymeis present in low amounts in the cell (T. Ny, Ph.D. thesis,University of Umea, 1979). Proteins expressed at low levelsusually do not show growth rate variation (26). For example,two other tRNA biosynthetic enzymes, tRNA (m'G)methyl-transferase and tRNA (mnm5s2U)methyltransferase, are pre-

* Corresponding author.

sent in low amounts in the cell, and the amounts of theseenzymes are invariant with the growth rate (14, 23, 25; T. G.Hagervall, Ph.D. thesis, University of Umea, 1984). Tounderstand the regulatory mechanism governing the synthe-sis of the tRNA (m5U)methyltransferase, it is necessary toestablish the genetic organization of its gene. The trmA gene,suggested to be the structural gene for this enzyme, islocated close to argH at 89 min and has been cloned (24).Here we present results indicating that (i) the trmA geneindeed codes for tRNA (m5U)methyltransferase, (ii) thetrmA gene constitutes a monocistronic operon, and (iii) thedirection of transcription is counterclockwise on the stan-dard E. coli K-12 chromosomal map.

MATERIALS AND METHODS

Abbreviations. Abbreviations used in this paper areaccording to the recommendations by the Commission onBiochemical Nomenclature (CBN-1983): the tRNA methyl-transferase producing m5U is called tRNA (m5U)methyl-transferase (EC 2.1.1.35). TrmA- and trmA designate thephenotype and genotype, respectively, of a mutant deficientin m5U in its tRNA. Apr, Tcr, and Cmr designate plas-mid-mediated resistance to ampicillin, tetracycline, andchloramphenicol, respectively. Neor designates coresistanceto both neomycin and kanamycin, and kb denotes kilobasepairs.

Strains and plasmids. The E. coli K-12 strains and plas-mids used are listed in Table 1.Media and growth conditions. The medium used was LB of

Bertani (3) supplemented with medium E (32), 0.2% glucose,and thiamine (1 ,ug/ml) unless otherwise stated. Medium wassolidified with 1.5% agar (LA plate). Antibiotics were used inLA plates at the following concentrations: ampicillin (100,ug/ml), tetracycline (20 ,ug/ml), kanamycin (50 ,xg/ml), strep-tomycin (100 ,ug/ml), and chloramphenicol (50 ,ug/ml).

Scoring of phenotype. For measurement of enzyme activ-ity, colonies were transferred into 0.5 ml of LB medium and

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TABLE 1. Bacterial strains and plasmids

Designation Sex Relevant markers or phenotype Referenceor source

Bacteria1100 F- Endol (9)M2141 F- A(lac-pro) rpsL minA minB (1)GB7 HfrP4x argH trmA5 thiA (24)GB802 F- argH trmA5 rpsL recA (24)

PlasmidspBR322 Apr Tcr (8)pTN102 Apr TCr TrmAr (24)pTNO15 Cel-' Imm(E1) TrmA+ (24)pSHI44 Cmr Tcr (20)pGP100 Apr TrmA+ This paperpGP1l1 Apr TrmA- This paperpGP102 Cel-' Imm(E1) Neor TrmA- This paperpGP103 Cel-' Imm(E1) Neor TrmA- This paperpGP104 Cel-' Imm(E1) Neor TrmA- This paperpGP105 Cel-1 Imm(E1) Neor TrmA- This paperpGP106 Cel-1 Imm(E1) Neor TrmA+ This paperpGP200 Apr Cmr TrmA- This paper

grown overnight. Cells were collected by centrifugation andsuspended in 50 ,ul of buffer A (10 mM potassium phosphate[pH 7.0], 0.1 mM dithiotreitol, 1 mM EDTA, 10% ethyleneglycol). The mixture was sonicated, and cell debris wasremoved by centrifugation. The resultant enzyme extractwas incubated for 1 to 2 h at 37°C in a 100-,ul reactionmixture containing tRNA from a trmA mutant as a substrate(22).

Plasmid DNA preparation. Plasmid DNA preparations andscreening of plasmids were by the methods of Edlund et al.(10) and John Collins (personal communication).

Restriction endonuclease analysis and ligation procedure.The conditions for restriction endonuclease digestion werethose suggested by the manufacturers (New EnglandBiolabs, Inc., Beverly, Mass.; Boehringer GmbH, Mann-heim, Federal Republic of Germany). Ligation was per-formed essentially according to the manual of BethesdaResearch Laboratories, Inc. (Gaithersburg, Md.). Analysesof plasmids and DNA fragments on agarose gels wereperformed as described by Edlund et al. (10).

Analysis of proteins expressed in minicells. The differentplasmid derivatives obtained from subcloning and TnS trans-positions were transformed into the minicell-producingstrain M2141. Preparation and labeling of plasmid-containingminicells were essentially as described by Kennedy et al.(15) and by Thompson and Achtman (31).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Radioactive minicell samples were analyzed as described byLaemmli and Favre (16), with the following modifications.The running gel was a gradient of 10 to 17.8% acrylamide and0.27 to 0.47% N,N'-methylene bisacrylamide. The stackinggel was a 5% acrylamide-bisacrylamide gel (30:0.8). Elec-trophoresis was performed until the bromphenol dye hadreached the bottom of the gel. In parallel to the radioactiveminicell samples, a low-molecular-weight standard sample,consisting of proteins ranging from 14.4 to 94.4K wasapplied. Gels were fixed in 45% methanol and 9% acetic acidfor 30 min and stained for 3 h in a solution containing 0.06%Coomassie blue G in 3% perchloric acid. After destaining in5% acetic acid, gels were dried and autoradiographed.

Preparation of transcriptional complexes. The standardassay mixture (50 ,ul), described previously by Stuber and

Bujard (29), contained the following (in 1 ml): 120 pumol ofKCI, 20 ,umol of Tris hydrochloride (pH 7.9), 10 ,umol ofMgCl2, 0.1 ,umol of EDTA, 0.2 pumol each of ATP, and GTP,0.05 ,umol each of CTP and UTP, and 1 to 5 jig of superheli-cal DNA. This mixture was preincubated for 1 min at 37°C,and transcription was started by the addition of RNA poly-merase at ratios of 2 to 4 enzyme molecules per promoterand then allowed to proceed for 0.5 to 5 min. Transcriptionwas stopped by diluting samples into a buffer suitable forrestriction endonuclease digestion containing streptolydiginat 100 ,ug/ml and an amount of restriction endonucleasesufficient to linearize the template DNA within 2 min at 37°C.The transcriptional complexes were prepared for electronmicroscopy as previously described (30).

Transformation and transposition. Transformation of plas-mid DNA was essentially as described by Mandel and Higa(19). The transposition experiment with TnS from Xkan (Ac1857 b221 rex::TnS, kindly given by H. Hennicke,Regensburg, Federal Republic of Germany) and strain GB7containing pTNO15 was performed as described elsewhere(7). Neor colonies were selected on minimal plates contain-ing medium E (32), 0.2% glucose, arginine (25 jig/ml),thiamine (1 j,g/ml), and kanamycin (50 jig/ml). The plasmidswere transferred to strain GB802 (recA trmA5) by F-mediated transfer, selecting for Arg+ Neor. Such cloneswere analyzed by restriction enzyme mapping, and clonescarrying TnS insertions in the 2.8-kb EcoRI fragment werechosen for further characterization.

Construction of trmA-cat gene fusions. Transcriptional fu-sions of the cat gene to the trmA promoter were obtained bypartial digestion of plasmid pGP100 with restriction endonu-clease TaqI followed by the addition of TaqI-digested pShI44(gift from S. Le Grice) and subsequent ligation of themixture. After transformation into strain 1100, Strr Apr Cmrclones with a TrmA- phenotype were found.

RESULTS

The trmA gene of E. coli K-12 has previously beenlocalized to a 2.8-kb E. coli DNA fragment 4kb clockwisefrom the argECBH operon (24). To map the control regionand determine the direction of transcription, several plasmidderivatives were constructed and analyzed by (i) expressionin minicells, (ii) insertion mutagenesis with transposon Tn5,(iii) in vitro transcription, and (iv) transcriptional fusion.

Subcloning of the trmA region and TnS insertion. The trmAgene was originally cloned in a ColEl hybrid plasmid,pTNO15, and subcloned into pBR322 as a 2.8-kb EcoRIfragment, pTN102 (24). The enzyme tRNA (m5U)methyl-transferase, purified to near homogeneity (12; Ny, Ph.D.thesis) comigrated with a 42K polypeptide expressed fromplasmids pTNO15 and pTN102 in minicells (24). To locate thestructural gene, plasmids pGP100 and pGP1l1 were con-structed (Fig. 1) and analyzed for the TrmA+ phenotype andexpression of [35S]methionine-labeled polypeptides inminicells. Plasmid pGP100, which contains a 1.4-kb EcoRI-PvuII fragment, confers the TrmA+ phenotype and in addi-tion specifies synthesis of a polypeptide comigrating with the42K protein of pTN102 (Fig. 2). Plasmid pGP1l1, carryingthe 1.0-kb EcoRI-SalI fragment, was TrmA- and specifiedno 42K polypeptide in minicells. Taken together, the aboveresults indicate that the 42K polypeptide is the trmA geneproduct tRNA (m5U)methyltransferase. In addition we con-clude that the structural gene is located in the leftward half ofthe 2.8-kb EcoRI fragment, between EcoRI and PvuII (Fig.1). After F-mediated transfer of TnS insertions in pTNO15,

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ORGANIZATION AND TRANSCRIPTION OF E. COLI trmA GENE

aSal I PVU

CdiEcoRl Sal I

pGPIOI4.7kb

EcoRlPyUI ori

PVd EooRIori

EcoRI 1 2 3

I . V

2

IRB

TAA TL(-1) (-2)

cda

Sal I pvuI

1kb

i

Sal I4 5

4

TAA(0)

FIG. 1. Subcloning of the trmA region and transcriptional fusion of the trmA promotor region to the structural gene of chloramphenicolacetyltransferase. Plasmid pTN102 (b) is a derivative of pBR322, containing a 2.8-kb chromosomal fragment with the trmA region insertedin the unique EcoRI site (ori denotes the replication region of pBR322) (8). Upon digestion of pTN102 with either Sall or PvuII ligation, andtransformation, plasmids pGP1l1 (a, SaiI) and pGP100 (c, PvuII) were obtained, carrying a 1.0-kb EcoRI-SalI fragment or a 1.4-kbEcoRI-PvuII fragment of the original insert, respectively. In (d) the TaqI sites within the enlarged EcoRI-PvuII chromosomal fragment ofpGP100 are numbered to simplify identification of the insertion point of the promoter free fragment of the cat gene schematically shown in(e), where R denotes the ribosomal binding site. Relevant stop codons for translation within the cat fragment are indicated; the numbers inparentheses define the fram of these triplets relative to the reading frame of the cat gene.

150 kanamycin resistant clones were scored for the TrmA-phenotype: 9 such clones were found. Four of these,pGP102, pGP103, pGP104, and pGP105, carrying the trans-poson at different sites within the putative trmA gene (Fig.3), were analyzed for the expression of [35S]methionine-labeled polypeptides in minicells. All four mutant plasmidsfailed to specify the 42K polypeptide identified as tRNA(m5U)methyltransferase (Fig. 2), indicating that the Tn5insertions are within the trmA structural gene, which againpositions the gene in a region corresponding to the EcoRI-PvuII fragment contained in pGP100. Upon insertion of TnS,truncated versions of the 42K protein showed up (smallarrows in Fig. 2). At least for pGP104 and pGP1O5, there isa good correlation between the size of these polypeptidesand the distance of the insertion points ofTnS to the leftwardEcoRI site in these plasmids (Fig. 3). Furthermore, a trun-cated polypeptide was synthesized from the plasmid harbor-ing the EcoRI-SalI fragment, pGP1l1 (Fig. 2). This suggeststhat the NH2 terminus of the tRNA (m5U)methyltransferaseis encoded in the immediate vicinity of the leftward EcoRIsite and that transcription in vivo proceeds from left to right.The plasmids pTNO15, pTN102, and pGP106 all synthe-

size the 23.5 and 24K polypeptides. However, since bothplasmids pGPlOQ and pGPlO fail to synthesize thesepolypeptides, their corresponding genes must be localized tothe right of the PvuII site and therefore downstream from thetrmA gene. In addition, none of the four TnS insertions in thetrmA gene was shown to affect the synthesis of the 23.5 and24K polypeptides. Since most, if not all, TnS insertionsaffect the expression of downstream genes in an operon,these results suggest that the genes for these polypeptidesare not cotranscribed with the trmA gene (Fig. 1 and 3).

Direction of transcription and mapping of the trmA controlregion. Plasmid pGP100 was chosen for the analysis of thetrmA control region because it contains the smallest frag-ment of the gene expressing the functional 42K protein. Inaddition, it is a pBR322 derivative, and pBR322-specifctranscriptional signals have been mapped and characterizedpreviously (29). The assay employs in vitro transcription ofcovalently closed circular DNA analyzed by electron mi-croscopy. Transcription was stopped at different time pointswith streptolydigin, and the DNA-RNA complexes werephotographed. Measurements of relative lengths and extrap-olation of the RNA length to possible starting points weremade. The compiled results from the population of mole-cules measured are shown in Fig. 4. Using the pBR322promoters P3 (bla), P4, and Pp for orientation of the pGPI00transcriptional complexes, an additional promoter (Pa) wasidentified at 35% of the genome length of pGP100 (Fig. 4). Painitiates RNA synthesis in the immediate vicinity of theEcoRI site within the 1.4-kb chromosomal insert in a direc-tion opposite to the ,B-lactamase gene and toward the PvuIIsite. Thus, we conclude that this promoter is the trmApromoter. Most likely, these transcripts are not terminatedwithin the cloned fragment, but within the pBR322 part ofthe plasmid pGP100 near the origin of replication. By com-paring the number of RNA chains initiated per time unit atPa and P3, it appears that, under our in vitro conditions,these promoters are equal in strength.The PvuII site is close to the Sail site, located within the

trmA gene (Fig. 1). Since the HindlIl site in Fig. 3 is locatedwithin the argECBH cluster (24), the direction of transcrip-tion of the trmA gene is toward argECBH and thus counter-clockwise on the standard E. coli K-12 chromosomal map.

EcoRI Sa Ib l l

C

d

EcoRI

e

Jmi pTN1027.0kb

---

pGPIOO3.7kb

--

Pvtl

I

lOObp

pGP2005.0kb

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1120 LINDSTROM ET AL.

C\j CN T- 0

CM o 0 0

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42K K-

30K-a24K

23.5K

(D Lo vt cv N LoO O cO OJ tl0 0 0 0 0

9- y- I- 9- 0QL 0- QL 0C Q- Z

Q00 H-

I1Ii11 1 1_ _ _ _ _ _~~~~~~~~~~~~~~~~~

VW w w w _

~~~~~-

_**r S_p _w a vw _

1*

24K

( 23.5K

FIG. 2. Electrophoretic analysis of [35S]methionine-labeled polypeptides from plasmid derivatives in minicells from M2141. Plasmidderivatives used are indicated with their names aligned to the respective lane. Lanes pTN102, pGP100, and pGP1l1 show expression fromplasmid constructions described in the legend to Fig. 1; lanes pGPI02, pGP103, pGP104, and pGP105 show expression from' Tn5 insertionsinto plasmid pTNO15 as outlined in Fig. 3. Plasmid pGP106 is a control plasmid with transposon TnS inserted outside the trmA region. Thepositions of the polypeptides made from plasmids pGlOl, pG104, and pGP105 are indicated by small arrows to the right of the resppective lines.Indicated with arrows are the 42K polypeptide [tRNA (m5U)methyltransferase] and the 23.5 and 24K polypeptides expressed from the 2.8-kbEcoRI fragment of plasmid pTN102. The 30K polypeptide is the P-lactamase of pBR322.

Psti Psti

pTNO15 LL'16.4kb

EcoRI

102103

EcoRI Sal I EcoRl HindIH Pati'I

EcoRI,, I

104Li

-105

pGP102 200 bp < 75 aa

pGP103 - 200 bp < 75 aa

pGP104

pGP105

0

700 bp < 245 aa

-_ 1100 bp < 365aa

500 1000 2800 bp/1

FIG. 3. Derivatives of pTNO15 carrying transposon Tn5. For plasmids pGP102, pGP103, pGP104, and pGP105 the insertion points oftransposon TnS within the 2.8-kb chromosomal fragment containing the trmA region are indicated by triangles, and arrows show the relativedirection of the transposons. In the lower part of the figure, bars represent the maximal coding capacity ofDNA between the EcoRI site andthe insertion points, taking into account that transposon TnS, independent of its orientation, contains stop codons in all reading frames withinthe first 30 base pairs (2). Abbreviations: bp, base pairs; aa, amino acids.

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ORGANIZATION AND TRANSCRIPTION OF E. COLI trmA GENE

Transcriptional fusion of the trmA promoter to the cat gene.Digestion of plasmid pSH144 with restriction endonucleaseTaqI yielded a 1,700-base-pair promoter-free fragment con-taihing the ribosomal binding ,site and the eptire structuralgene for chloramphenicol acetyltransferase (17, 18). UponcloAing of this fragment into. pGP100 partially digested byTaqI, four Apr Cmr TrmA clones were found and subse-quently analyzed with respect to the site of insertion and thebrientation of the cat gene and assayed in the minicellsystem for expression of plasmid-coded polypeptides. Allfour clones carred the cat ene inserted in the structural genefor tRNA (m5U)methyltransferase in a direction opposite tothe gene for P-lActamdsae (Fig. 1), suggesting that in vivo thetrmA gene in pGPlI) is transcribed from left to right. In

:t

Pvul EcoRI PvuI EcoRI PvuNl_

t§X I I I Ii f0 0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1

Fractional bngth

FIG. 4. Analysis of transcriptional complexes of plasmidpGP100. After formation of transcriptional complexes (30), plasmidpGP100 was linearized with restriction endonuclease PvufI. Eachpoint in the dot plot in the upper part of the figure shows the position(along the x axis) and the length (along the y axis). For bettervisualization, the length of the synthesized RNA molecules wasdoubled, and each point is represented twice both above and belowthe line (30). The position and direction of mapped transcripts areschematically represented by arrows in the lower part of the figure.The plasmid is drawn as a double molecule to visualize transcriptspassing over the PvuII site. Pa, P3, P4, and Pp indicate thetrmA,P-lactamase, P4, and Pp promoters, respectively. The numbers ofmolecules analyzed were too few to determine the position of P4. Itslocation is therefore taken from Stuber and Bujard (29).

A 27KB

42K

30K

FIG. 5. Densitometric comparison of relative expression inminicells from plasmids pGP100 and pGP200. (A) Pattern fromplasmid pGP100, where the 42 and 30K polypeptides indicate theposition of tRNA (m5U)methyltransferase and P-lactamase, respec-tively. (B) Relative expression from the fusion plasmid pGP200where the 27 and 30K polypeptides represent chloramphenicolacetyltransferase and j-lactamage, respectively. The 17K polypep-tide is a possible truncated tRNA (m5U)methyltransferase as dis-cussed in the text.

minicells, not only Was the 27K chloramphenicol acetyl-transferase visible, but also truncated versions of the 42Kprotein were observed (Fig. 5). Taking into account theknown termination codons within the cat fragment (20), thesizes of these truncated proteins locate the NH2 terminus ofthe tRNA (m5U)methyltransferase in the immediate vicinityof the EcoRI site in pGP100 (Fig. 1).

DISCUSSION

The results presented in this paper suggest that the struc-tural gene for tRNA (m5U)methyltransferase (trmA) consti-tutes a monocistronic operon. Subclonings established thatthe trmA gene is localized to the leftward 1.4-kb EcoRI-PvuII fragment of plasmid pTN102 (Fig. 1). Since tRNA(m5U)methyltransferase has an Mr of 42,000, it requiresabout 1.1 kb of DNA for its transcription and translation.Transcription in vitro demonstrated that the promoter (Pa) isclose to the EcoRI site and the direction of transcription istoward the PvuII site (Fig. 4). This conclusion is supportedby the appearance of truncated polypeptides from plasmidshaving TnS insertions at different positions within the trmAgene as well as plasmid pGPlOl (Fig. 2). This conclusion i!also confirmed by the appearance of an additional 17Kpolypeptide expressed from the cat fusion plasmid pGP200(Fig. 1 and 2). Formation of this polypeptide is most likelydue to termination of translatioji at one of the two stopcodons proximal to the NH2 terminus of the cat gene (Fig.1), since the predicted size of such a truncated trmrA geneproduct is approximately 17K. The 23.5 and 24kpolypeptides are encoded from the rightmost half of the2.8-kb EcoRI fragment (Fig. 1) and thus downstream fromthe trmA gene. All TnS insertions may not be polar withrespect to downstream genes in polycistronic operons. HoW-ever, none of the four Tn5 insertions within the trmA geneaffected the synthesis of the 23.5 and 24K polypeptides inminicells. Three of the insertions (103, 104, and 105) have thesame orientation, whereas insertion 102 shows the oppositeorientation. These results suggest, but do not prove, that theexpression of the 23.5 and 24K polypeptides is not part of thetrmA transcription unit.

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The suggested start site of the trmA mRNA close to theEcoRI site might be an internal promoter that is activatedupon subcloning. Recently, the btuB gene was sequenced(13). The direction of transcription of the btuB gene isopposite to that of the trmA gene. The promoter regions ofthe btuB gene and the trmA are separated by about 200 basepairs, and no additional promoterlike sequence can be foundin this region (unpublished results). Therefore we suggest thepromoter close to the EcoRI site to be the trmA promoterused in vivo.We have purified the tRNA (m5U)methyltransferase and

estimated that the amount present in the cell at a specificgrowth rate of 1.0 is below 100 molecules per genomeequivalent (T. Ny, Ph.D. thesis). This is about the same levelas the tRNA (mtG)methyltransferase, a tRNA-modifyingenzyme that has been estimated to be present at about 80molecules per genome equivalent (14). Comparing this witha level of ribosomal proteins of about 8,000 and aminoacyltRNA synthetases of about 500 to 1,000 molecules pergenome equivalent and only 20 molecules for a repressorprotein, such as if the proposed downregulation was on thetranscriptional level, we expected a rather weak promoter invitro. However, by calculating the number of initiations pertime unit in Fig. 4, it is apparent that the trmA and blapromoters initiate transcription equally well in vitro. The blapromoter has previously been shown to be of averagestrength in pBR322 (29). If these promoters are assumed toreflect an average E. coli promoter efficiency, the trmApromoter is moderately strong in vitro. Expression inminicells also equals that of the bla gene irrespective ofwhether the polypeptides were labeled with [35i]methionineor a 14C-amino acid mixture (data not shown). Taken to-gether, these results suggest that regulation of the trmA geneis mainly on the transcriptional level. Since our results ontranscription and translation are from in vitro experimentsand minicells, we cannot exclude the possibility of additionalregulatory mechanisms acting on the trmA gene in vivo.

ACKNOWLEDGMENTSThis work was supported by the Swedish Cancer Society (project

no. 680), the National Science Foundation (BU-2930), the SwedishBoard of Technical Development, and the Deutsche Forschungsge-meinschaft (Bu 338/12). Part of the work reported in this paper wasinitiated during an EMBO short-term fellowship to P.L.

Kerstin Kjellin-Straby, Stuart Le Grice, and Hermann Bujard aregratefully acknowledged for support and stimulating discussions.

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2. Auerswald, E.-A., G. Ludwig, and H. Schaller. 1981. Structuralanalysis of TnS. Cold Spring Harbor Symp. Quant. Biol.45:107-113.

3. Bertani, G. 1951. Studies of lysogenesis. I. The mode of phageliberation by lysogenic Escherichia coli. J. Bacteriol.62:293-300.

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5. Bjork, G. R. 1984. Modified nucleosides in RNA-their formationand function, p. 291-330. In D. Apirion (ed.), Processing ofRNA. CRC Press, Inc., Boca Raton, Fla.

6. Bjork, G. R., and L. A. Isaksson. 1970. Isolation of mutants ofEscherichia coli lacking 5-methyl uracil in transfer ribonucleicacid or 1-methyl guanine in ribosomal RNA. J. Mol. Biol.

51:83-100.7. Bjork, G. R., and A Olsen. 1979. A method for isolation of

Escherichia coli mutants with aberrant RNA methylation usingtranslocatable drug resistance elements. Acta Chem. Scand.Sect. B 33:591-593.

8. Bolivar, F., R. L. Rodrigues, P. J. Greene, M. C. Betlach, H. L.Heyneker, and H. W. Boyer. 1977. Construction and character-ization of new cloning vehicles. II. A multipurpose cloningsystem. Gene 2:95-113.

9. Durwald, H., and H. Hoffman-Berling. 1968. Endonucleasel-deficient and ribonucleaseI-deficient Escherichia coli mutants.J. Mol. Biol. 34:331-346.

10. Edlund, T., T. Grundstrom, and S. Normark. 1979. Isolationand characterization of DNA repetitions carrying the chromo-somal P-lactamase gene of Escherichia coli K-12. Mol. Gen.Genet. 173:115-125.

11. Gegenheimer, P., and D. Apirion. 1981. Processing of procary-otic ribonucleic acid. Microbiol. Rev. 45:502-541.

12. Greenberg, R., and B. Dudock. 1980. Isolation and character-ization of m5U-methyltransferase from Escherichia coli. J. Biol.Chem. 255:8296-8302.

13. Heller, K., and R. J. Kadner. 1985. Nucleotide sequence of thegene for the vitamin B12 receptor protein in the other membraneof Escherichia coli. J. Bacteriol. 161:904-908.

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