bacteriophage t5 transfer rna · 2001-09-05 · in procedure b, gel electrophoresis for the first...

Bacteriophage T5 Transfer RNA ISOLATION AND CHARACTERIZATION OF tRNA SPECIES AND REFINEMENT OF THE tRNA GENE MAP**

(Received for publication, October 23, 1979)

Clayton Hunt,g Suresh M. Desai, Judith Vaughan, and Samuel B. Weiss From IThe Franklin McLean Memorial Research Institute, The Departments of Biochemistry a n d Microbiology, and The Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637

Previous studies from this laboratory have provided a high resolution map for 16 tRNA genes located on the continuous heavy DNA strand of bacteriophage T5 DNA (Chen, M.-J., Locker, J., and Weiss, S. B. (1976) J. Biol. Chem 251, 536-547). All of the T5 tRNA genes were located in three clusters within the DNA C segment, except for tRNAA", which mapped on the left end of the DNA D segment (Desai, S. M., Hunt, C., Locker, J., and Weiss, S. B. (1978) J. Biol. Chem 253, 6544- 6550). In this report, we present evidence for the presence of eight additional T5 tRNA species, five of which are located in two new loci within the DNA C segment. We also describe a two-dimensional gel electrophoresis system for the separation and isolation of T5 tRNA species from crude infected RNA preparations. The gel electrophoresis system separates tRNA isoacceptors specific for different amino acids; evidence is presented that the isoacceptors for isoleucine, histidine, and serine are coded by different T5 genes.

The presence of T5-coded tRNA molecules in T5-infected extracts from Escherichia coli has been reported previously (1-3). Although the function of T5 tRNAs in phage develop- ment is still unclear, we have attempted to map the tRNA genes in the T5 genome with the expectation that such a map eventually would be useful in studies of transcriptional control of tRNA synthesis. As a result of this effort, several tRNA isoacceptors have been identified which map in different regions of the T5 genome (4, 5). A high resolution map (Fig. 1) for 16 of the T5 tRNA genes has been constructed on the basis of electron microscopic heteroduplex analysis of T5 deletion mutants, coupled with information obtained from tRNA- DNA hybridization data (5).

Fig. 1 (upper diagram) depicts the entire T5 DNA molecule (approximately 80 X 10" daltons) with the four major single strand interruptions that divide the light strand into five unequal segments designated as A, B, C, D, and E (6, 7). The

* This study was supported in part by the National Institutes of Health Grant AI-12479, and is taken, in part, from a Ph.D. thesis submitted by C. H. to the Graduate School of The University of Chicago. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solelv to indicate this fact.

+Dedicated to Elwood V. Jensen on the occasion of his 60th birthday. 3 Supported by a National Research Service Award, GM 07183

(Public Health Service National Institute of General Medical Sci- ences) and by a United States Public Health Service Training Grant T32 CA 09183 (National Cancer Institute). I The Franklin McLean Memorial Research Institute is operated

by The University of Chicago for the United States Department of Energy under Contract No. EY-76-C-02-0069.

map of Fig. 1 (lower diagram) shows that all of the genes for the T5 tRNA species examined thus far are present in the C segment of the heavy T5 DNA strand except for tRNAArK, which is located at the left end of the DNA D segment (8). In this report, we present 1) evidence for the presence of five additional T5 tRNAs and for the isoacceptors of tRNAH'" and tRNASe', and 2) a description of the two-dimensional gel electrophoresis and chromatographic procedures used for the isolation and identification of individual T5 tRNA species.

MATERIALS AND METHODS

Growth a n d Purification of T5 Phages and Isolation of Phage DNA-Wild type T5' and all the T5 deletion mutants were grown and purified, and phage DNA was isolated, as described by Chen et al. (5).

Isolation of tRNA from Phage-infected Cells-The conditions for phage infection were essentially as described previously (5). E. coli F, grown on NCG medium (8 g of nutrient broth (Difco), 10 g of casamino acids (Difco), 20 g of glycerol, 2.5 ml of 1 M Tris-HC1, pH 7.8, in a total volume of 1 liter) to a density of 3 to 4 X IO8 cells/ml a t 37°C in a rotary shaker, was infected with T5 phage at a multiplicity of 10 following the addition of CaCL at a final concentration of 1 mM. Fourteen minutes later, chloramphenicol (50 pg/ml) was added and incubation continued for 60 min. Cells were harvested by centrifuga- tion, and crude tRNA fraction was isolated by phenol extraction and chromatography on Whatman DEAE-52, as reported elsewhere (4). T5 r3'P]tRNA was prepared in the same manner, except for the following modifications. E. coli F was grown in low phosphate medium (approximately 0.1 mM phosphate) as described by Pinkerton et al. (9), and carrier-free '*PI (25 mCi/70 ml of culture medium) was added 8 min before the addition of chloramphenicol (8).

Preparation of T5-specific tRNAs by Hybridization-The crude infected tRNA preparation (described above) was used for the isolation of T5-specific tRNAs by annealing to denatured T 5 phage DNA, and the hybridized RNA was recovered as reported elsewhere (10).

Aminoacyl Synthetase and tRNA Charging-Aminoacyl synthetase was prepared from soluble cell extracts of E . coli MRE-600 by a procedure reported previously (2). Aminoacylations of tRNA with "S-, "C-, or "H-labeled amino acids (New England Nuclear) were carried out under conditions described elsewhere (5).

Reversed Phase Chromatography-Transfer RNA, charged with a single radioactive amino acid or labeled with '"P, was subjected to chromatography in the RPC-5 system of Pearson et al. ( l l ) , under conditions previously reported (4).

Two-Dimensional Gel Electrophoresis-'"P-Labeled T5 tRNA preparations were subjected to two-dimensional acrylamide (acrylamide/N,N"methylenebisacrylamide, 20:l) gel electrophoresis by the procedure of Ikemura and Dahlberg (12). Two separate gel conditions were employed. In Procedure A, the first dimension was a 10% gel polymerized in 0.5 X TEB buffer (1 X TEB contained 21.6 g of Tris base, 1.86 g of Na'EDTA, and 11 g of boric acid/liter, adjusted to a final pH of 8.3) containing 0.4% 3-dimethylaminopropionitrile and 0.1% ammonium persulfate. This gel was formed in a vertical electrophoresis apparatus (10 x 20 cm) (E-C Apparatus Corp., St. Peters- burg, Fla.), cooled to 13°C by a circulating water bath, and run at 400 V in 0.5 X TEB buffer until the bromphenol blue marker dye had migrated about 18 cm. A gel slice containing the appropriate ["'PIRNA bands was removed, placed in a second vertical E-C appa-

3 164

by guest on October 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

The Gene Map of T5 tRNAs

T!? Phooe DNA

H 5 ' A B C D E 3'

L 3' 3 0 3.8 5 1 13.9 1 4 . 5 5' "

tRNA Gene Mop

I II m m A lo Pro

H IS , Ser V a l I le , ,Gly Leu

I l e L Phe

Asp, Met Lys TY r Arg Metf

2 . 9 6 k b l 0 3 k b 0 . 3 9 k b 1 . 0 3 k b

b 3 i I i i+ I I I

I I I b 4 I -

I I

b 2 I I

b l i

st(14) I 1 - 1

ratus (20 X 20 cm) at right angles to the first dimension run, and a 20% acrylamide solution containing 0.33% 3-dimethylaminopropionitrile, 0.5 X TEB buffer, and 0.07% ammonium persulfate was polymerized around the first dimension gel slice. Electrophoresis in the second dimension was for 18 h at 400 V in 0.5 X TEB buffer a t 13°C.

In Procedure B, gel electrophoresis for the first dimension was in 15% acrylamide containing 0.3% 3-dimethylaminopropionitrile, 0.5 X TEB buffer, and 0.125% ammonium persulfate. The gel was formed in a modified E-C electrophoresis apparatus (10 X 40 em) and was run at 800 V for 18 h in 0.5 X TEB buffer at 13OC. The second dimension gel solution contained 20% acrylamide, 4 M urea, 0.19% 3- dimethylaminopropionitrile, 1 X TEB, and 0.07% ammonium persulfate. This solution was polymerized around an appropriate gel slice containing [32P]RNA bands from the fist dimension run in a modified E-C electrophoresis apparatus (20 X 40 cm) and was run at 800 V for 60 h in 1 X TEE buffer at 13°C.

Following electrophoresis, the labeled RNA spots were located by autoradiography, excised from the gel, ground with a glass rod in a small vial, and extracted by incubation with 2 to 3 ml of 0.3 M NaCl for 2 h at 37°C. The extract was collected by filtration through a Millipore filter (0.45 p ) , and the ["'PIRNA was concentrated either by addition of 50 pg of carrier yeast RNA (periodate-treated) and ethanol precipitation, or by adsorption onto small DEAE-52 columns (2 to 3 mm in height in a disposable pipette) and elution with 0.8 M NaCl in a final volume of 0.3 to 0.4 ml. Carrier RNA (15 pg) was usually added to the DEAE-eluate, followed by ethanol precipitation. The two- dimensional gel RNA extracts were used for hybridization analysis, RPC-5 chromatography, aminoacyl charging, and fingerprint analysis.

Fingerprint Analysis of fZP]RNA-Two-dimensional fingerprints of ['"PJRNA were obtained by digestion either with T 1 or pancreatic RNase (Worthington) according to the methods developed by Sanger and his collaborators (13, 14). Following nuclease treatment, the labeled sample was applied to cellulose acetate strips and electrophoresed for 45 min at 5000 V in pH 3.5 buffer (5% glacial acetic acid, 0.5% pyridine, 0.002 M EDTA). The partially separated oligonucleotides were transferred to DE81 paper (Whatman) by blotting and were electrophoresed perpendicular to the first dimension in 7%

6 .28

4.49

6 I O

7.79

I O 02

3165

FIG. 1. Physical and tRNA gene map of T5' phage DNA. Upper diagram, the schematic representation of T5' DNA was constructed from previously reported models (6,7) as described by Chen et al. (5). The heavy continuous and the light DNA strands are indicated as H and L, respectively. The letters A, E , C , D, and E designate the DNA segments produced by the major nicks in the light strand; the approximate molec- ular weights (X of each light strand DNA segment are given by the numbers below each segment. Lower diagram, the tRNA gene map and the map position of the mutant T5 DNA deletions were derived from previous reports (5, 8). The top bar represents the complete C segment of T5' DNA, with the B-C and C-D single strand interruptions shown by the discontinuous space. The lower bars represent the positions of the DNA segments deleted in the eight mutant phages listed on the left. The sizes of the deletions (kilobases) are listed on the right. The vertlcal broken lines define the four regions of T5' DNA (loci I to IV) containing tRNA genes as speci- fied by the amino acids listed under each region. The numbers above the brackets represent the kilobase length of the individual tDNA regions; the numbers be- neath the top bar give the kilobase length for the spaces between Regions I- 11, 11-111, and 111-IV, as well as the dis- tance from the B-C nick to the left end of Region I.

formic acid a t IO00 V for 4 h. Oligonucleotides were detected by autoradiography.

Modified Base Analysis-Analysis for major and minor nucleotides contained in various T 5 [32P]RNA species was carried out by means of the two-dimensional thin layer chromatography (TLC) method of Saneyoshi et al. (15). The labeled RNA was digested with T2 RNase (Sankyo) in 0.05 M potassium acetate (pH 4.7) and then applied to cellulose TLC chromatography plates (20 X 20 cm) (Brink- man). The first dimension was developed with isobutyric acid, 0.5 M NH,OH (53, v/v) and the second dimension with isopropyl alcohol/ concentrated HCI/HOH (70:15:15, v/v/v). Nucleotides were detected by autoradiography and identified by co-migration with known stand- ards (where available) or by comparison with the reported chromatographic properties of specific minor nucleotides (15).

RESULTS

Detection and Mapping of T5 tRNA Glutamic Acid, Tryp- tophan, Asparagine, Threonine, Cysteine, and Glutamine- Six T5 tRNA species (glutamic acid, tryptophan, asparagine, threonine, cysteine, and glutamine) are absent in the gene map of Fig. 1. Our earlier studies had indicated the presence of T5 tRNA"'" in infected E. coli extracts (5), but we encoun- tered difficulties in determining its gene location; the synthesis of the other five tRNA species was uncertain. Table I shows that charging of T5-infected cell tRNAs with radioactive tryptophan, asparagine, threonine, cysteine, and glutamine, followed by annealing to T5 DNA, gives positive hybridization for each "H-aminoacyl-tRNA, indicating that these tRNA species are coded by the T5 genome. Table I1 summarizes the hybridization data obtained when the six nonmapped ,"H- aminoacyl-tRNA species were annealed with the various T5 mutant DNAs. The charged tRNA for glutamine, cysteine, tryptophan, and asparagine showed hybridization with only


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3166 The Gene Map of T5 tRNAs

TABLE I Hybridization of T5‘-infected 3H-aminoacyl-tRNAs to T5’ DNA Transfer RNA prepared from T5”infected cells was aminoacylated

with a single labeled amino acid and annealed with denatured T5’ DNA fixed on nitrocellulose fdtefs in 50% formamide 0.03 M sodium citrate, pH 6.0 (final volume, 0.5 ml), in the absence and presence of 200 pg of E. coli tRNA, under identical conditions for each charged tRNA. Each reaction mixture contained a second filter of denatured T4 DNA as a control. The amino acids were labeled with tritium, except for cysteine which was labeled with .”“S, with specific activities that ranged from 2.9 to 21 Ci/mM. The total input radioactivity in the individual annealing mixtures ranged from 2 X 10‘ to 2 X lo5 cpm. Following annealing, the hybridized radioactivity was determined as described elsewhere (5) and normalized to counts per min per 40 pg of DNA.

Total counts fixed to filters

cpm 1439 1524 3966 3985 695 918

7405 635 1 59 1 652

CPm 97 78

484 432 86

133 2979 2507

9 5

cPm 1342 1446 3482 3553 609 785

4426 3844 582 647

TABLE I1 Hybridization of labeled T5’ 3H-aminoacyl-tRNAs to T5 mutant

DNAs T5”infected cell tRNA was individually charged with the six

radioactive amino acids shown below, annealed with wild type and mutant T 5 DNAs, and the counts found in the hybrid complex determined as in Table I. The conditions for the annealing reaction were the same as Table I, except that the final annealing volume was 3.0 ml. Hybrid counts, after correction for control counts bound to blank filters, were considered positive if they were within 35% of wild type values. The labeled amino acids used for charging were the same as in Table I except for F’Hlplutamic acid, specific activitv 22 Ci/mM.

Arnino- DNA

tRNA T5’ b l b2 b3 b4 st(0) st(8) st(14) st(20) acyl-

Glu + ” ” ” - + c y s + - - - - - - - + Trp + - - - - - - - + Asn + - - - - - - - + Gin + - + + + + + + + Thr + - + + + + + + +

one mutant DNA, st(20), suggesting that these four tRNA genes are contained within a common deletion sequence which is delineated by the left end of the st(8) deletion and the right end of the st(14) deletion (Fig. 1). T5 tRNA”’” and tRNAThr hybridize to all deletion DNAs except bl; hence, their gene location must be to the right of the left end of the bl deletion and to the left of either the b2 or b4 deletion, which places them in Region I of the T5 DNA C segment already known to contain six tRNA genes.

Gel Electrophoresis of tRNAs by Procedure A-The purification of individual tRNA species is essential for determi- nation of the presence of tRNA isoacceptors, for restriction endonuclease mapping, and for sequence analysis. Shortly after T5 infection of E. coli (3 to 4 min), shut-off of host RNA synthesis occurs (16), which permits the specific labeling of T5 RNA transcripts by the addition of ‘*Pi to the infected media. Since “’P has a short half-life and the purification of individual tRNA molecules is relatively lengthy by conven- tional chromatographic techniques, we examined gel electro-

phoresis as a procedure for rapid isolation of individual T5 tRNA species.

Initially, T5-specific labeled RNAs were prepared from phage deletion mutants expected to code for relatively few tRNA species. Fig. 2 illustrates the autoradiograms of crude [““PIRNAs prepared from T5 bl-, st(14)-, and b3-infected cells following two-dimensional gel electrophoresis by Procedure A, as described under “Materials and Methods.” The bl deletion mutant, expected to code only for tRNAAru (Fig. l) , showed one major and one minor radioactive spot, designated as 1 and 10-11, respectively (Fig. 2 A ) . The st(14) and b3 deletions demonstrated a larger number of labeled RNA spots which were similar in position except for the additional b3 spot, 13 (Fig. 2, B and 0. Occasionally, the complex Spots 1- 2 and 7-8 were partially resolved by the electrophoresis procedure, and an additional spot (X) was sometimes seen (Fig. 2 0 . When the 1-2 spot of st(14) and b3 was extracted and electrophoresed in 16% acrylamide-containing 7 M urea, three separate bands (1,2, and 3) appeared (Fig. 20).

To establish the amino acid specificity of tRNAs associated with each radioactive area, we excised individual gel spots, extracted and concentrated the RNA, and examined it for charging with different “H-amino-acids. The gel-extracted RNAs which showed positive charging were subjected to hybridization with T5’ DNA to insure that the “H-aminoacylated tRNAs were of phage origin. The results from such chargings and hybridizations are indicated by the amino acid assignments for the T5 tRNA species next to each numbered spot and band (in brackets) in Fig. 2. RNA isolated from radioactive Spots 10-11 and 12 (Fig. 2) demonstrated no

r 10 x

T5 bl

10-11(7) - IArd

spot 1-2

~~

FIG. 2. Separation of T5 tRNA species by two-dimensional polyacrylamide gel electrophoresis according to Procedure A. The preparation of T 5 ‘”P-labeled tRNAs, and the conditions for electrophoresis by Procedure A, were as described under “Materials and Methods.” Panels A, B, and C show the autoradiographic spots obtained following electrophoresis of [:‘ZP]RNAs isolated from cells infected with the T5 phage mutants bl, st(14), and b3, respectively. Electrophoresis in the fmt dimension (right to left) was in 10% polyacrylamide; the second dimension electrophoresis (top to bottom) was in 20% polyacrylamide. The labeled RNA from each gel spot was extracted and charged with different “H-amino-acids. Those RNA extracts showing positive charging with one or several “H-amino- acids, and positive hybridization to T5’ DNA, are identified by the amino acid(s) in brackets next to each numbered spot. Panel D shows the separation of the T5 tRNA species glutamine, glycine, and arginine when the RNA extract of Spot 1-2 was subjected to a thud dimension electrophoresis in 16% polyacrylamide containing 7 M urea.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

The Gene Map of T5 tRNAs 3167

detectable charging with any labeled amino acid, were devoid of modified bases, and gave fingerprint patterns unlike any of the T5 tRNA species described in this report.

Chromatography of Electrophoretic-separated tRNAs- Further identification of the above labeled T5 ["'PItRNA species separated by gel electrophoresis was established by reversed phase chromatography. Figs. 3 and 4 illustrate the RPC-5 profiles obtained for 10 of the T5 ["'PItRNAs isolated by two-dimensional gel electrophoresis and co-chromatographed with T5-specific tRNAs charged with their putative "H-amino-acids. Qualitatively, the coincidence of the double

Fraction Number FIG. 3. RPC-5 chromatography of T5 tRNA species isolated

by two-dimensional gel electrophoresis. The T5 32P-labeled RNAs extracted from the gel spots of Fig. 2 were individually cochro- matographed on RPC-5 with T5-specific tRNAs (free from E. coli tRNA) charged with a single %amino-acid. The RPC-5 profiles for the distribution of 3zP and 3H radioactivity are shown above. The labeled RNA material co-chromatographed in each of the above panels was as follows: A, Band 1 [32P]RNA derived from Spot 1-2 (Fig. 2 0 ) plus T5 [3H]glutaminyl-tRNA; B, Band 2 ["PIRNA derived from Spot 1-2 (Fig. 2 0 ) plus T5 [3H]glycyl-tRNA; C, Band 3 ["'PIRNA derived from Spot 1-2 (Fig. 2 0 ) plus T5 ["Hlarginyl-tRNA; 0, Spot 7 ["2P]RNA (Fig. 2 B ) plus T5 ["Hlmethionyl-tRNA; and E, Spot 8 ["'PlRNA (Fig. 2 B ) plus T5 ['Hlthreonyl-tRNA.

Fraction Number FIG. 4. RPC-5 chromatography of T5 tRNA species isolated

by two-dimensional gel electrophoresis. The conditions and labeled RNA materials subjected to co-chromatography on RPC-5 were similar to those described for Fig. 3, except for the following: A, Spot 3 r3'P]RNA (Fig. 2 B ) plus T5 [3H]isoleucyl-tRNA; B, Spot 4 r3*P]RNA (Fig. 2 B ) plus T5 ['HH]histidyl-tRNA; C, Spot 9 ["'PIRNA (Fig. 2 B ) plus T5 [3H]histidyl-tRNA; D, Spot 6 rY2P]RNA (Fig. 2C) plus T5 [3H]seryl-tRNA; and E, Spot 13 [32P]RNA plus T5 ["Hlseryl- tRNA.

labeled profiles was generally good for most of the RPC-5 runs. This result supports the amino acid assignments for the T5 [32P]RNAs isolated by gel electrophoresis; nevertheless, slight variations were observed in several cases. Fig. 3A indicates that Band 1 [32P]RNA (derived from Spot 1-2, Fig. 2 0 ) and T5 [3H]glutaminyl-tRNA both gave two labeled peaks; however, the double labeled profiles showed some discordance and were not precisely coincident. Variations of this type could arise from differences in base modifications of tRNA species, especially since the two T5 tRNA preparations were derived from cells grown under different conditions: The T5 [32P]RNAs were prepared from infected cells grown in low phosphate medium, whereas the T5 tRNAs used for charging were isolated from infected cells grown in NCG medium


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3 168 The Gene Map of T5 tRNAs

containing excess phosphate. Chromatographic variations of this type have been observed for E. coli tRNAs grown under different phosphate concentrations (17).

The ['"PIRNAs isolated from Bands 2 and 3 showed very good coincidence with the multiple isoacceptor peaks found with the "H-aminoacylated tRNA species for glycine and arginine, respectively (Fig. 3, B and C). As mentioned earlier, the conditions used for gel electrophoresis only partially resolved Spots 7 and 8; thus, the labeled material isolated from these two spots was invariably cross-contaminated. Fig. 3 0 indicates that only the fvst ['"PIRNA peak of Spot 7 is coincident with T5 ["H]methionyl-tRNA, whereas the second ['"'PIRNA peak of Spot 8 chromatographs like the major peak of T5 ["Hlthreonyl-tRNA (Fig. 3E).

Spot 3 [,'"P]RNA from the st(14) mutant gave two radioactive peaks on RPC-5 which were precisely coincident with the two peaks derived from T5' tRNA charged with [''HI- isoleucine (Fig. 4A). However, Spot 3 [:"P]RNA from the b3 mutant chromatographed as a single peak coincident only with the first [:'H]isoleucyl-tRNA peak (data not shown). These observations are consistent with our previous findings which indicate that T5' DNA contains two tRNA"" genes, one of which (Ile?) is deleted in all the T5 mutants except st(20) and st(14) (4).

T5' [:'H]histidyl-tRNA gives four prominent peaks on RPC- 5; however, the [:"P]RNA from Spot 4 shows coincidence with Peaks 2, 3, and 4 (Fig. 4B), whereas the labeled RNA from Spot 9 is coincident with Peak 1 (Fig. 4C). Similarly, T5' ["Hlseryl-tRNA demonstrates six radioactive peaks on RPC- 5; the ['"PIRNA from Spot 6 chromatographs with Peaks 3.5, and 6 (Fig. 40) , whereas the labeled RNA from Spot 13 is associated with Peaks 1 and 4 (Fig. 4E). These data suggest the presence of multiple tRNA genes for histidine and serine. No ['"PIRNA from any of the labeled st(14) or b3 mutant gel spots was found to chromatograph with the T5' ["Hlseryl- tRNA isoacceptor of Peak 2.

Fingerprint Analysis of RPC-5 Isoacceptor tRNAs-The multiple RPC-5 peaks obtained with each labeled tRNA isolated by gel electrophoresis were subjected to fingerprint analysis for assessment of similarities and differences in nu-

Gln peak l

T

e

e '* 0

e

P 0

T

0 0 . 'I

9 .

P 0

GIy peak 1

.

T

. *

I

cleotide sequence arrangement for these chromatographic isoacceptors. Figs. 5 and 6 show that the multiple ["?P]RNA peaks coincident with the "H-aminoacyl-tRNA peaks for glutamine, glycine, and arginine (Fig. 3, A, B, and 0 exhibit similar T 1 and pancreatic RNase fingerprint patterns (for each separate tRNA species), suggesting a common nucleotide sequence arrangement for the chromatographic isoacceptors that have the same amino acid specificity. However, somewhat different results were obtained for the RPC-5 isoacceptors of isoleucine, histidine, and serine.

Fig. 7 shows that the fingerprints for Peaks 1 and 2 of tRNA"" (Fig. 4A, Spot 3) are distinctly different; this is

r.

r

f e ,

0

P

A r q oear 3

e 0

c h

a

0

FIG. 6. Fingerprint analysis of the chromatographic i tRNA speciesfor arginine. The three RPC-5 isoacceptor peaks for [."P]tRNAArY (Fig. 3C) were subjected to fingerprint analysis as described for Fig. 5.

Glg peok 2

. * . . . ' 1 . ..

0

T

P I

.-

. . b.

I..

FIG. 5. Fingerprint analysis of the chromatographic T5 tRNA species for glutamine and glycine. The two

tRNA"'" and ["I']tRNA';" (Fig. 3, A and HPC-5 isoacceptor peaks for [:"PI-

B ) were subjected to fingerprint analysis following TI and pancreatic RNase digestion as described under "Materials and Methods." The autoradiograms of the RNase digest of the ["'PltRNA isoacceptors, followed by two-dimensional electrophoresis, are shown above. Elec- trophoresis was on Whatman DE81 paper, right to left in the first dimension and top to bottom in the second dimension. The letters T and P (TI and pancreatic RNase, respectively) at the bottom of each panel indicate the nuclease used for digestion. The letter B in each fingerprint shows the position of the xy- lene cyano1 dye.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

The Gene Map of T5 tRNAs

Ile peak 1 I l e peak 2 M e t Thr

3169

['"PItHNA FIG. 8. 1

HIS, peak 2 HIS, peak 3 Hlsl peak 4

0 .

0 .E

0

#

P 0

r

0 b o

0 .

.E

8

0

P .

a

0

P 0

FIG. 7. Fingerprint analysis of the chromatographic T5 tRNA species for isoleucine, methionine, and threonine. The two HI'C-5 isoacceptor peaks for ['"']RNA"'' (Fig. 4 A ) and the major HPC-5 peaks for [~"I']tHNA~"' and ['"I']tKNA""" (Fig. 3 , D and h') were subjected to fingerprint analysis as described in Fig. 5.

' 0

I)

peaks for

consistent with our previous evidence for the presence of two that of Peak 1 tRNA'"" (Fig. 4C. Spot 9, His2). This informa- different tRNA"' genes in T5' DNA (4). Fig. 8 indicates that tion reinforces the suggestion that T5 DNA contains two the fingerprints of Peaks 2, 3, and 4 of His (Fig. 4B, Spot 4, different tRNA'"" genes (His, and His2), and that three of the His,) are similar to each other, but distinctly different from four T5' tRNA"'" isoacceptor peaks (2,3, and 4) seen on RPC-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3170 The Gene Map of T5 tRNAs Ser, peak 3 Ser, peak 5

T T

B

P

Ser, peak 6

. .'+, .-

' 0

T

' 0

P

SerZ peak 1

FIG. (Fig. 4,

9. Fingerprint analysis of the chromatographic T5 tRNA species for serine. D and E ) were subjected to fingerprint analysis as described in Fig. 5.

0

0

0 eo

0

c

~

@ 511.91

ij

Serz peak 4

- *

. .. .

0

T

The five RPC-5 isoacceptor peaks for [ "P]tRNA*"

FIG. 10. Separation of T5 tRNA species by two-dimensional polyacrylamide gel electrophoresis according to Procedure B. The preparation of T5 '"P-labeled tHNAs and the conditions for electrophoresis by Proce- dure B were as described under "Mate- rials and Methods." Panels A and C show the autoradiograms obtained following electrophoresis of ["'PIRNAs isolated from cells infected with T5' wild type and T5 st(8) phage, respectively. Electrophoresis in the fmt dimension (right to left) was in 15% polyacrylamide; that in the second dimension (top to bottom) was in 20% polyacrylamide containing 4 m urea. Panels B and D are schematic drawings of the autoradiograms for T5' and st(8) ["'PIRNAs, respectively. The amino acid shown next to a radioactive spot indicates the specificity of the T5 tRNA contained in that gel spot which was determined as described in Fig. 2, and by fingerprint comparison with those tRNAs identified by Procedure A (Fig. 2).

5 most probably represent modification variants of one of the the T5 tRNASer species isolated from Spots 6 and 13 by gel gene products (His,). In Fig. 9, similar fingerprints are ob- electrophoresis are coded by different tRNA genes (in agree- served for the ["*P]RNA associated with RPC-5 Peaks 3, 5, ment with Henckes et al. (3)), but that the chromatographic and 6 of T5' tRNA%' (Fig. 4 0 , Spot 6, Ser,); however, the isoacceptor peaks detected for each of these gene transcripts fingerprint patterns of Peaks 1 and 4 (Fig. 4E, Spot 13, Ser2), represent modification variants of a single gene product. although similar to each other, are different from the isoac- Gel Electrophoresis of tRNAs by Procedure B-For wild ceptors of Spot 6 (Sen). These results support the idea that type T5' and several phage deletion mutants which produce


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


a larger number of T5 tRNAs than st(14) and b3, the two- TABLE 111 dimensional gel electrophoresis procedure described in Fig. 2 Identification of T5 tRNA species by electrophoresis for T5’ and was inadequate for resolving certain tRNA species. Modifi- T5phage mutant-infected cells cation of the conditions for gel electrophoresis described in ‘”P-Labeled tRNA was prepared from E. coli F cells infected with Procedure B (see “Materials and Methods”) increased the T5’ and the T5 phage mutants shown below, as described under

resolution of T5 tRNA species and allowed the separation of “Materials and Methods”. The labeled preparations of T5 tRNA were subjected to two-dimensional gel electrophoresis by either Procedure

autoradiograms obtained for J‘P-labeled RNAs prepared from charging with “H-amino-acids, hybridization to T5 I)NA, and finger- T5’- and st(8)-infected cells, respectively, when these are print analysis.

more tRNA mixtures. Fig. 10 ( A and c) shows the A or Procedure B (“Materials and Methods”) and identified by

subjected to electrophoresis by Procedure B. Fig. 10 (B and -tRNA spe-. T5, st(20) st(8) st(o) b3 st(,4) b4 hl D) contains schematic drawings of the radioactive areas ob- a e s

”

served in the above autoradiograms in which certain spots His, have been given an amino acid assignment based on aminoacyl His? charging and fingerprint analysis of the isolated RNAs. Sev- era1 gel spots have the same amino acid assignment, indicating Ilel Ser?

that the RNAs isolated from these areas charged with the ~l~ same amino acid. The gel spots with the same amino acid Thr designation without subscript numbers (e.g. there are five Met,

Ala Pro

9

Leu e Phe . - - B

0

T

+ + + + + + + - + + + + + + + - + + + + + + + - + + + + + - + - + + + + + + + - + + + + + + + - + + + + + + + - + + + + + + + - + + + + - + + + + - ”

- - - -

proline spots) give similar T1 and pancreatic RNase fingerprints, whereas spots having the same amino acid assignment, but different subscript numbers (eg. Ser, and Serd show distinctly different fingerprint patterns. Gel spots that have no amino acid assignment represent T5 [:I2P]RNAs which have not yet been identified and may or may not contain functional tRNA species. Fig. 11 illustrates the T1 and pancreatic RNase fingerprints for the T5 tRNA species isolated and identified in Fig. 10, in addition to those shown in Figs. 5 to 9.

Production of tRNAs by T5 Deletion Mutants-Table I11 is a summary of the T5 tRNAs identified by two-dimensional gel electrophoresis following phage infection of E. coli with T5’ wild type and the various T5 deletion mutants.

DISCUSSION

The results presented here refine and extend our earlier reports on the detection and map location of T5 tRNA genes. In this study, the use of reversed phase chromatography and electrophoresis for the purification of T5 tRNA species gave rise to the detection of tRNA isoacceptors (11, 18-20). For mapping purposes, it was important to determine whether these isoaccepting species represented base modification variants of a single gene product or whether they were transcribed from unique genes. To distinguish between these two types of isoacceptors, we used fingerprint analysis which relies on the specificity of T1 and pancreatic ribonuclease to cleave RNA

FIG. 11. Fingerprint analysis of the T5 tRNA species isolated chains aiselect purine or pyrimidine nucleotide sites (13, 14).

by ewo-dimensional gel electrophoresis (Procedure B). The T5 For most of the isoaccepting T5 tRNA species examined, the [np]RNAs isolated by two-dimensional gel electrophoresis, Proce- fingerprint Pattern were remarkably similar, suggesting a dure B (Ficc. 101. were fmeemrinted as described under “Materials common primary structure. Nucleotide analysis showed some and Methouds.” The finge$ri& for the radioactive gel spots assoc i - differences in modified bases.’ On the other hand, the chro- ated with a specific tRNA species and identified Leu, Phe, Ala, matographic and electrophoretic isoacceptors for T5 Lys, Pro, met, Asn, and Trp are shown here. The fingerprints for the tRNAHi% Ile. Ser

other T5 tRNA species identified, but not shown (Gln, Gly, Arg, Ilel, showed distinctly different fingerprints, sug-

Ile2, Met.,,,, Thr, His,, Hh, Ser,, and Ser2), were the same as shown in I C. Hunt, S. M. Desai, J. Vaughan. and S. B. Weiss, unpublished Figs. 5 to 9. results.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3172 The Gene Map of T5 tRNAs

r ma m b ma m b

Ala

His,, HlsZ Pro Phe Gln, Serz Val Asn Ile,, Gly Leu Cys Thr, Met, L y s Glu Ile2

ASP Metf Trp Tyr Ser, Arg 2 . 9 6 kb 1.03kb 1.78kb 0.39kb 2.26kb 1.03kb

I I

5.28 kb I Sf(2Ot I

I I

s t ( 8 ) I I l

S t ( 0 ) I

!

T 5 * $ ! I C 1 1 1 I I j D f I '

b 3 I I : I I I I I

I 1 I I I I

b 4 I I I

I I I

I

s t (14) I 1 - 1

I b 2

b i - gesting the presence of at least two structurally different tRNAs recognized for each of these species.

The detection of five additional T5 tRNA species and the ability to monitor the production of mutant phage tRNAs by gel electrophoresis have added confirmation to our earlier tRNA gene map and contributed to its refinement, as illus- trated in Fig. 12. The tRNA genes located in Region I are shown to be Hisl, Hisz, Gln, Sers, Ilel, Gly, Thr, Mef, and Asp. The presence of two histidine isoacceptor genes is sug- gested by the different fingerprint patterns obtained for the two tRNAHis spots isolated by electrophoresis (Fig. 8). In addition, the two tRNAHi" species show hybridization to two different restriction endonuclease fragments of T5 DNA.' Both tRNA"'" species are produced by all T5 deletion mutants except b l (Table 111); thus, their genes are located in Region I, in agreement with our previous hybridization results (5).

Analysis of T5 tRNA species synthesized by phage mutants indicates that tRNA;'" is produced by all of the T5 mutants except b l and st(14) (Table 111). The absence of Serz synthesis by st(14) is inconsistent with its production by both the b3 and b4 mutants, whose DNA deletions span the st(14) deletion. If we disregard the lack of Serz production by st(14) (see discussion below), then Ser, maps to the left of the b4 deletion and to the right of the left end of the b l deletion, which define the boundaries of Region I.

The tRNA species Gln, Ilel, Gly, Thr, and Met, show hybridization to all the T5 mutant DNAs except b l (Table I1 and our earlier studies (5)), and are produced in cells infected with each mutant phage except b l (Table 111). Hence, these tRNA genes are also located in Region I of the map. The gene for tRNAA"" is positioned in Region I based on our previous findings (5).

The tRNA genes located in Region I1 of Fig. 12 have not been altered from their previous map position (Fig. 1). The data of Table 111 support our original map location for five of the Region I1 genes (Ala, Pro, Leu, Lys, and Metf), since these tRNA species are produced in infected E. coli cells only by those phage mutants (st(20), st(@, and st(0)) whose deletions do not encompass the 1.03 kb DNA segment of Region 11.

Region IIIa of the T5 DNA C segment represents a new

I I

FIG. 12. The revised physical map for T5 tRNA genes. This map is similar to that shown in Fig. 1, but, in addition, shows the approximate DNA location for eight new T5 tRNA species, i.e. Hisz, Gln, Thr, Asn, Cys, Glu, Trp, and Sen. The position for the gene tRNAPhe has been relocated from Region IIIb to IIIa; Regions IIIa and IVa are new tRNA gene loci in T5 DNA.

locus for tRNA genes which was not previously identified. The location of the genes for asparagine, cysteine, glutamic acid, and tryptophan in this new region stems from the annealing data of Table 11, which show hybridization for these tRNA species only to one mutant phage DNA, st(20). Thus, the above genes are positioned in a 1.78 kb length of the DNA C segment defined by the left end of the st(8) deletion and the right end of the st(14) deletion. The data of Table I11 agree with the map location for asparagine, cysteine, and tryptophan, since the production of these tRNA species in infected cells was found only with the st(20) mutant; all other mutants delete this gene region. The production of tRNA"'" by the T5 mutants is uncertain since it has not been identified following gel electrophoresis.

We had reported earlier that tRNA''h" hybridized poorly to st(14) DNA (only 25% that of T5+ DNA) and not at all to b4 DNA (5). Our present studies indicate that, among all of the T5 mutants examined, the st(20) mutant is the only one that expresses the phenylalanine gene, similar to the genes for asparagine, cysteine, and tryptophan (Table 111). Based on tRNA production and the marginal hybridization to st(14) DNA, the gene for tRNAPhe has been relocated to Region IIIa of the T5 DNA map (Fig. 12).

The physical map positions for the tRNA genes Iles and tyrosine in T5' DNA remain unaltered from their previous location in the 0.39 kb DNA segment designated as Region IIIb in Fig. 12 (5). Fingerprint analysis of the two tRNA"' species isolated (Ilel and Ile2) supports the presence of two different isoleucine tRNA isoacceptor genes in T5+ DNA; the production of tRNA2"' only by st(20) and st(14) (Table 111) is in agreement with its map location. The tRNA"Yr gene product has not been identified following gel electrophoresis of crude T5 tRNAs.

The gene for Serl has been mapped in a previously unoc- cupied locus, Region IVa. The production of tRNA,"" in infected cells was detected with the mutants b3, st(14), and b4, but not with any of the other T5 deletion mutants (Table 111). In addition, annealing of "'P-labeled tRNA'"" with T5' segments whose ends correspond to the position of the T5 DNA nicks in the light strand (8) shows hybridization to the


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


DNA C segment, but not to the D segment.' These observations suggest that the Serl gene is located in a 2.26 kb DNA length of the C segment that extends from the right end of the b3 deletion to the C-D nick, which delineates the boundaries of Region IVa. The absence of tRNA1'"' production of the bl mutant suggests some discrepancy in the mapping or size of the bl, or the b3 deletion, or both, since the b3 deletion (as mapped) extends further to the right than the b l deletion (Fig. 12). The location of the tRNAArR gene at the left end of the T5+ DNA D segment (Region IVb) was derived from previous work and has not been altered (8).

Certain discrepancies between the production of specific tRNA species by several DNA deletion mutants and the map position for these deletions have been mentioned above. The mutant deletions were positioned in the T5 genome by heteroduplex mapping and the measurement of heteroduplex contour lengths by electron microscopy was reported to contain an error of 5 to 10% (5). Between any two deletion mutants, the relative map position of the DNA deletion segments could be in error by 10 to 20%. Since the DNA deletion boundaries define the tRNA gene regions, these regions may be in error to the same extent. Inaccurate positioning of DNA deletions in the T5 map could account for the unexpected appearance or lack of production of a specific tRNA species by a particular T5 deletion mutant. In addition, the presence of unidentified small deletions (100 to 200 nucleotides in length) in transcriptional control elements or in structural genes, not observed by heteroduplex mapping (5), could also account for the absence of an expected RNA product.

Our present study indicates that T5 DNA codes for at least one tRNA species specific for each of the 20 amino acids utilized in protein synthesis. The total number of individual T5 tRNA genes thus far identified is 24, which is far larger than the number of tRNAs produced by the T-even phages (1, 18, 21). BF23, a phage closely related to T5, has been reported to produce some 20 different RNA spots by two- dimensional gel electrophoresis which migrate like tRNAs (22). The complexity of the tRNA species produced by T5 is somewhat surprising in view of its relatively small genome size (80 X lo6 daltons) and especially since the tRNA genes appear not to be required for phage growth.

Acknowledgments-We wish to acknowledge the kind assistance of Doctors J. E. Dahlberg and E. Lund at the University of Wisconsin, Madison, Wisc., who instructed us on the use of gel electrophoresis and fingerprint analysis and generously offered the use of their laboratory facilities.

REFERENCES

1. Scherberg, N. H., and Weiss, S. B. (1970) Proc. Natl. Acad. Sci.

2. Chen, M.-J., Shiau, R. J., Hwang, L-T., Vaughan, J., and Weiss,

3. Henckes, G., Panoyatakis, O., and Heyman, T. (1976) J. Virol.

4. Hunt, C., Hwang, L-T., and Weiss, S. B. (1976) J . Virol. 20,63-69 5. Chen, M-J., Locker, J., and Weiss, S. B. (1976) J. Biol. Chem.

6. Hendrickson, H. E., and Bujard, H. (1973) Eur. J. Biochem. 33,

7. Hayward, S. D., and Smith, M. G. (1973) J. Mol. B i d 80, 345-

8. Desai, S. M., Hunt, C., Locker, J., and Weiss, S. B. (1978) J. Biol.

9. Pinkerton, T. C., Paddock, G., and Abelson, J. (1973) J . Biol.

10. Scherberg, N. H., and Weiss, S. B. (1972) Proc. Natl. Acad. Sci.

11. Pearson, R. L., Weiss, J. F., and Kelrners, A. D. (1971) Biochim.

12. Ikemura, T., and Dahlberg, J. E. (1973) J. Biol. Chem. 248,5024-

13. Sanger, F., Brownlee, G. G., and Barrell, B. G. (1965) J. Mol. Biol.

14. Brownlee, G. G., and Sanger, F. (1967) J . Mol. Biol. 23, 337-353 15. Saneyoshi, M., Ohashi, Z., Harada, F., and Nishimura, S. (1972)

16. Lanni, Y. T., and McCorquodale, D. J. (1963) Virology 19, 72-80 17. Mann, M. B., and Huang, P. C. (1974) J. Bacteriol. 118, 209-212 18. Desai, S. M., and Weiss, S. B. (1977) J. Biol. Chem. 252, 4935-

19. Kelmers, A. D., Novelli, G. D., and Stulberg, M. P. (1965) J . Biol.

20. Fradin, A., Gruhl, H., and Feldmann, H. (1975) FEBS Lett. 50,

21. McClain, W. H., Guthrie, C., and Barrell, B. G. (1972) Proc. Natl.

22. Ikemura, T., and Ozeki, H. (1975) Eur. J. Biochem. 51, 117-127

U. S. A. 67, 1164-1171

S. B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,558-562

17, 316-325

251, 536-547

529-534

359

Chem. 253,6544-6550

Chem. 248,6348-6365

U. S. A. 69, 1114-1118

Biophys. Acta 228, 770-774

5032

13,373-398

Biochim. Biophys. Acta 262, 1-10

494 1

Chem. 240,3979-3983

185-189

Acad. Sei. U. S. A. 69,3703-3707


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

C Hunt, S M Desai, J Vaughan and S B Weissand refinement of the tRNA gene map.

Bacteriophage T5 transfer RNA. Isolation and characterization of tRNA species

1980, 255:3164-3173.J. Biol. Chem.

http://www.jbc.org/content/255/7/3164Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/255/7/3164.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/255/7/3164

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;255/7/3164&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/255/7/3164

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=255/7/3164&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/255/7/3164

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/255/7/3164.full.html#ref-list-1

http://www.jbc.org/

bacteriophage t5 transfer rna · 2001-09-05 · in procedure b, gel electrophoresis for the first...

Documents