Structure of a yeast non-initiating methionine-tRNA gene Judith

volume 8 Number 91980 Nucleic Ac ids Research

Structure of a yeast non-initiating methionine-tRNA gene

Judith Olah+ and Horst Feldmann

lnstitut fur Physiologische Chemie, Physikalische Biochemie und Zellbiologie der UniversitStMunchen, Goethestrasse 33, 8000 Munchen 2, GFR

Received 17 March 1980

ABSTRACT

4 to 8 kb Hind III fragments of yeast DNA were cloned intopBR322. One of these clones (pY6m3) containing a single tRNA#et

gene has been characterized in detail. The DNA sequence of thestructural gene is colinear with the tRNA sequence, which meansthat in this case no intervening sequence is present. The 5'-leader and 3'-trailer sequences have also been determined. The5'-flanking region can be folded up into possible secondarystructures.

INTRODUCTION

The spectrum of tRNA precursors obtained by in vivo pulse

labelling of processing deficient mutants [1-3] suggests that

there are two classes of tRNA genes in yeast: some genes that

do contain intervening sequences, others that do not. So far,

mainly those precursors to yeast tRNA species [ 3-6 ] or the cor-

responding genes [7,8] have been, analyzed, which have an intron

next to the anticodon. in vitro transcription and processing of

these tRNAs have been studied in some detail [3,9-lll. On the

other hand, there have been relatively few characterizations of

the 5'-leader and 3'-trailer sequences of in vivo yeast precur-

sor tRNAs and of their processing. Also, the published yeast

tDNA sequences do not extend very far into the flanking regions.

As we are interested in the possible clustering of yeast

tRNA genes, we have begun a study of such genes that are con-

tained in relatively large cloned yeast DNA fragments. We have

isolated a number of plasmids which contain at least three tRNA

genes (J. Olah and H. Feldmann, unpublished) and which are ana-

lyzed at present. Another set of plasmids was obtained which

carry genes for tRNA^6 [ 12]; here we wish to report on the

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Nucleic Acids Research

analysis of one of these genes.

ARTERIAL AND METHODS

Chemicals. [ p]phosphate (carrier-free) was purchased from12 5

New England Nuclear Corp. Na I, high specific activity, was

obtained from the Radiochemical Centre, Amersham. SeaKem and

Seaplaque agarose were products of Marine Colloids, Rockland.

Acrylamide and N.N1-Bismethylenacrylaraide were bought from

Serva, Heidelberg. Other chemicals were of analytical grade.

Total yeast tRNA was obtained from Boehringer GmbH, Mannheim;

tMBV^e and tRNA.,e (yeast) were prepared according to ref. 13;

other individual yeast tRWV species were a gift of G. Dirheimer

and G. Keith, Strasbourg. T4 DNA ligase was a product of Miles

Corporation. T4 polynucleotide kinase was from Boehringer GmbH,

Mannheim. Restriction endonucleases Hind III and Taq YI were

from Microbiological Research Establishment, Porton; Eco RI,

Sau 3A, Sau 96, Alu I, and Pvu II were gifts of R.E. Streeck;

Bam HI, Sal I, and Hae III were gifts of U. H&nggi.

Preparation of I1 l]tRKAs. tRIRs were labelled with * I by

a procedure [14] similar to ref. 15: 0.1 to O.2 A2g0-units of a

purified tRNA species (or 1 to 2 A-,--units of bulk yeast tRNA)

were combined with 2 ul 2M Na-acetate buffer, pH 5.0, 2 ul

O.625M KI solution, ca. 2 mci Na125I solution and 2 Ml O.O25M

Tlcl3 solution to a final volume of 20 ul in a micro reaction

tube. Incubation was carried out at 6O°C for 30 min. The sample

was then extracted with an equal volume of chloroform twice,

2 ul 0.2M Na.S-Oc solution added and ethanol precipitated. The

pelleted tRNA was dissolved in 20 ul of loading buffer and run

on a 1O% polyacrylamide gel as described earlier [16], tRNA

bands in the gel were localized by staining it with ethidium

bromide. The tRNA material was eluted from minced gel slices

directly into 2 ml of the hybridization buffer (see below). Spe-7 8

cific activities of the tRNAs were ca. 1O to 10 dpm/ug.

Yeast DN& (S. cerevisiae, C836) was prepared as described

earlier [17 1 and digested with Hind III. 1OO A.,^-units of the

phenoled digest were loaded onto a cylindrical Seaplaque

agarose gel (2.5 x 20 cm, 0.8%) in the following buffer: O.O4 M

Tris-acetate, O.O2 M sodium-acetate, 1 mM EDTA, pH 7.4 [ 18']. The

1976



gel was set up in a special device which allowed to elute DNA

fractions during electrophoresis at the bottom into the same

buffer. Electrophoresis was carried out for 20 hrs at 15O volts

and ca. 1OO fractions (4 ml) were collected. The DNA from these

samples could be directly recovered by ethanol precipitation.

Yeast DNA Hind III fragments were cloned using pBR322 as a

vector and E. coli K12 strain 49OA as a host [19]. Both

were obtained from R.E. Streeck. Ligation [ 20]and transformation

[21] conditions were as reported in the references. The cloning

experiments were carried out under P3/EK1 conditions according

to the NIH Guidelines, and after February 13, 1979, individual

characterized clones were grown under L2/B1 conditions according

to the German "Richtlinien zum Schutz vor Gefahren durch in-vitro

neukombinierte DNA". Appropriate clones were tested by the

colony hybridization technique [ 22] using [ 1] tRNAs as probes.

Hybridization was done overnight in 6xSSC, 5O% formamide [23],

Agarose gel electrophoreses were performed on 3 mm slab gels

using the buffer system as described in ref. 18. The gels were

stained with ethidium bromide and photographed according to

ref. 24. For subsequent hybridization with [ iltRNA the DNA

was transferred to nitrocellulose filters [25], which were in-

cubated in polyethylene envelopes in 6xSSC, 50% formamide at

56°C with 0.2 to 1 ml of tRNA solution (see above).

DNA fragments for sequencing were prepared by digestion with

the appropriate restriction enzymes and gel electrophoresis on

tube gels (13 cm 0) using Seaplaque agarose and the buffer

system described in ref. 18. Single bands were cut out of these

gels, the gel material melted at 7O°C and poured onto cylindri-

cal 10% polyacrylamide gels (0.08 M Tris borate buffer, pH 8.4,

1 mM EDTA). After several hours electrophoresis at 2OO volts,

the DNA had concentrated in this second gel as a sharp band.

Extraction, labelling of the DNA, and all other procedures for

sequencing were performed according to Maxam and Gilbert [26].

0.5 x 20 x 40 cm 8% and 2O% polyacrylamide slab gels were used.

Five different cleavage reactions were used; partial cleavage at

purine residues was achieved by the reaction described by Grayet al. [ 271.

1977



RESULTS AND DISCUSSION

1. Isolation and identification of plasmids carrying tRNA genes.

Hind III restricted S. cerevisiae DNA was fractionated by

preparative 0.8% agarose gel electrophoresis. An aliquot from

each fraction eluted from the gel was run out on 0.8% agarose

slab gels, the DNA transferred to nitrocellulose filters by the

Southern method [25] and the filters hybridized to [125l]tRNA"et

125and I-labelled unfractionated yeast tRNA. Several fractions

which contained DNA ranging in size from ca. 4 to 8 kb and which

hybridized to either tRNA, or to the total tRNA, were chosen

for cloning in the Hind III site of pBR322.

Ampicillin resistant and tetracycline sensitive colonies

arising from transfection of E. coli 490A by yeast DNA ligated

to pBR322 were screened by the colony hybridization technique

[22] for tRNA genes using total tRNA, and then several purified

tRNA species labelled with I. Altogether, 42 clones carrying

tRNA genes were isolated. One clone, designated pY6m3 hybri-

dizing to tRNA.,6 was chosen for a detailed analysis which is

discussed in the following.

2. Hind III, Eco RI and Hae III restriction enzyme analysis

of the hybrid plasmid from clone pY6m3.

Plasmid DNA was prepared from clone pY6m3 and the DNA cut

with Hind III, Eco RI, Hae III, and combinations of these en-

zymes and run out on O.6% agarose gels. The results are shown in

Fig. 1. Digestion of pY6m3 DNA with Hind III yielded the ex-

pected 4362 bp pBR322 DNA fragment and two additional DNA frag-

ments of length 3350 and 2540 bp. Since the original DNA frag-

ments used for cloning was ca. 6 kb large, this result suggests

that the population of DNA fragments obtained by Hind III

cleavage of yeast DNA contained fragments from partial di-

gestion.

To locate the gene onto the various restriction fragments,

the DNA in the agarose gels was transferred to nitrocellulose

filters [25] and hybridized to [125l]tRNA"et. As seen in Fig. 1Metthe tRNA, gene was assigned to the 3350 bp Hind III fragment,

a 2 3OO bp Eco RI fragment, and the largest fragments of the

Hae III (2OOO bp), the Hae III x Eco RI (18OO bp) and the

1978



aC

ma

XX

i—iCLOU

00)oIX

mC—

MetFiq.l : Restriction patterns of pY6m3 and location of the tRNA3

gene by hybridization.The standard is a mixture of linear Xdvl, Xdv21/Hind III,and Xdvl/Hae III [28].

Hae III x Hind III (2000 bp) digests.

To order the restriction sites relative to each other, DNA

fragments from digestion with one enzyme were eluted from

agarose gels and cut with the other enzymes. The resultant

restriction map is shown in Pig. 2A. Sites for Bam HI or Sal I

were not detected in the yeast DNA insert.

Met gene on the 180Q bp3. Fine structure mapping of the tRNA_, -- - -

Hae III x Eco RI DNA fragment.MetIn order to more precisely locate the tRNA. gene and

determine the number of copies, plasmid pY6m3 DNA was digested

with Hae III and then with Eco RI, analyzed on 0.8% agarose gels

and the 18OO bp fragment eluted from the gel. The DNA was then

restricted with various restriction enzymes producing shorter

fragments, fractionated on 1.5% or 2.0% agarose gels, blotted

and the filters again hybridized to [ I]tRNA M e t (not shown) .

The resultant fine structure restriction map is in Fig. 2B.

1979



Hind inB

1f

3 <O

3.0I I • I I

Kb

Fig.2: Restriction endonuclease cleavage map of pY6m3.A: The whole plasmid. Fragments are numbered accordingto size; pBR322 is drawn in black; the indicated seg-ment is the Hae III x Eco RI fragment containing thegene» — » . direction of transcription.Bt Fine structure map of the Hae III x Eco RI fragment.

From the published sequence of the tRNA molecule [ 12], two

Taq YI sites and one Alu I site within the gene were expected,

and this was confirmed. Only one Taq YI fragment (650 bp) and

one Alu I fragment (290-30O bp) hybridized to [125I]tRlft"et.

Since in the overlapping part of these two fragments the Alu I

site is to the left of the Taq YI site it is unlikely that

there is more than one copy of the tRNA., gene within the

650 bp Taq YI fragment. Supporting evidence for this comes

from the Sau 3A and Sau 96 restriction patterns. Only the

largest Sau 3A fragment (660 bp) hybridized with the probe and

this fragment has only about 1OO extra base pairs to the left

of the beginning of the tRNA gene. Similarly, only the largest

Sau 96 fragment (7 90 bp) hybridized with the tRNA probe and

this fragment has only about 100 extra base pairs on the

3'-side of the gene. From these results we conclude that within

the 59OO bp yeast DNA insert in plaamid pY6m3 only one copy ofMet

the tRNA 3 gene occurs.

1980



4. Sequences of the tRNAffet gene and its flanking regions.

We next used the Maxam-Gilbert technique [ 26 ] to determine

the sequence of the cloned tRNA gene and the sequences flanking

it. The strategy for sequencing is indicated in Fig. 2B; an

example of a sequencing gel is shown in Fig. 3, and the complete

sequence is documented in Fig. 4A. We should note that

sequencing in this case was more complicated than for yeast

Fig.3: Autoradiogram of a^ ^ ^ sequencing gel of the

O < o o < transcribed strandcontaining part ofthe structural geneand the 5'-flankingregion.

G

T

A

T

T

A

40G

A

C

1981


C » I C I T C G T C T A j T A A A T T T A I Q T T Q C T Q [ A ~ T A A A T T A f J T C T C C A T T Q J T T C T T T T A T T Q \ * * T A T T | A A | A O C A T T T A A T O C T J A a A A T C C T C C A T A A C A O * 1 AC t i C l l O C i C I T 1 * _ T _ T T * A A T | C A A C Q A C | l A T T T A A T A J A O A O O T A A C A A A O A A A A T A A C [ T _ T _ _ A _ T _ A _ A | T T [ T C O T A A A T T A C Q A | T C T T A G O A G G T A T I C T C » A l

I* m7 m5 m1 C C A

A_U A A V C U G A A Q O D C O A O A G T V C O A A C C U C U C C U O C A G C A

tO 50 60 70

1 T

C A T T T T T T T C A A A A C T T T T T A T C A C T Q C T A O T A A A O a T A A T O A A T O A O O A C O T T A C T O T A a T A C A A C C A C A C A TC T A A A A A A A O T T T T Q A A A A A T A Q T Q A C G A T C A T T T C C A T T A C T T A C T C C T Q C A A T Q A C A T C A T G T T f i G T C T G T A

o_CD

o'>od.u>3)CD(A

a>

B QCT T

T aa A

-90 * jOA T C T T C O T C T

T *

T - 7 0

* 0T T AT T A-

t '*9 TAT C T C C A T T G T T T C T T T T

A * TT T AA A TA A TT C

TT A

T A• A T

11TAA AOAATCCTCCATAACAGATAG

r A

8?TA

A

C TA CA A

ca

OC T

T T T ' ATO T A

Q A -30 • AT7.-70 CO

CTAQAAGCAQA AQAOOTAACA AOOAOOTATTOTCTATC

A C ,A A

T ?!

MetFig.4: Nucleotide sequence of a yeast tRNA^*" gene and adjacent regions.A: The sequence is shown in three blocks comprising the 5'-flanking region, thestructural gene, and the 3'-flanking region. The tRNA sequence is shown above thestructural gene. Symmetries in the 5'-flanking region are indicated by boxes; theclusters in the 3'-flanking region are underlined.B and C: Possible secondary structures within the 5'-flanking region.



tRNA ^ r [7 ] or tRNA [ 8 ] genes where sequencing could start at

an Xma I or an Eco RI site, respectively, located close to the

3'-end of the gene. Our results confirmed the sequence of the

tRNA- molecule, as previously determined [ 12 ], and established

the orientation of the tRNA gene relative to the Hind III site

of pBR322. in addition it was found that in this case the EKA

sequence is colinear with the tRNA sequence - in contrast to a

number of other tRNA genes in yeast which contain inserts next

to the anticodon [3,5,8 ]. The 31(C)CA-end is not encoded in the

DNA, as observed for other yeast tRNAs [7,8]. Obvious is the

relatively low (GfC) content within the flanking regions of this

gene, a fact that has been observed earlier [ 29 ] and found for

other yeast tRNA genes I 7,8 I. The 3'-flanking region has 37%

(G+C), there occurring a stretch of seven Ts close to the end of

the mature tRNA molecule. This was also found in a number of

other genes transcribed by yeast RNA-polymerase III and has been

considered a transcription termination signal [7,8,3O,31j. The

M£t

sequence ahead of the tRNA" gene has only 26% (G+C) content.

Upon comparison with 5'-flanking regions of other cloned tRNA

genes or the yeast 5S RNA gene [7,8,30,31] no obvious sequence

similarities could be detected. However, computer analysis re-

vealed that in our case this portion of the sequence (in con-

trast to the 3'-flanking region) possesses a high degree of

symmetry. Fig. 4A points out for example that there are three

shorter palindromes consisting of A and T only and one 12 bp

palindrome. Moreover, the 5'-flanking region due to these

symmetries can be folded up into hypothetical secondary struc-

tures as shown in Fig. 4B. We have to await the outcome of

further analyses, which we are pursuing at present, before we

can attribute any significance to these observations with regard

to promoter or processing sites. There is preliminary evidence

that this tRNA-6 gene is transcribed (in vitro), because in a

homologous transcription system (H. Feldmann, unpublished) we

observed a ca. 1OO nucleotides long pre-tRNA molecule.

5. Occurrence of other tRNA genes in the pY6m3 plasmid and

distribution of tRNA- and other tRNA genes.

We have attempted to find other tRNA genes on the 5900 bp

1983



yeast DNA insert. No hybridization was observed with the

following 125I-labelled pure species: tRNAMet, tRNAPhe,

tRNAAsp, tRNAAla, tRNA^al, and two tRNAsPr . Therefore, we have

hybridized the Eco RI x Hae III fragments and separately the

Sau 3A x Sau 96 fragments of the largest Eco RI x Hae III frag-

ment (compare Figs. 1 and 2) to total [ l]tRNA (experimentsMet

not shown). Since only the fragments containing the tRNA.

gene hybridized, we conclude that no other tRNA genes than this

are present in pY6m3. The same situation seems to apply to three

other plasmids similar in size to pY6m3 which have not yet been

further analyzed. Their DNA hybridized to tRNA-6 only, among

the purified tRNA species mentioned above; in comparison toMetother clones, all tRNA- gene carrying clones gave much weaker

autoradiographic signals after hybridization with total tRNA.

On the other hand, we have isolated a number of plasmids

which contain at least three tRNA genes in 5 to 8 kb large yeast

DNA inserts. Two out of these, that are being characterized more

closely at present, have the tRNA genes (for Asp, Ala, Val, and

Pro) on subfragraents s£ 18OO bp. The analysis of other yeast

tRNA genes and our observations suggest that there may be two

types of tRNA gene arrangement in yeast: dispersed genes seem to

occur for t R N A ^ ( 7 1, tRNAPhe [ 8 ] and probably for tRNAMet; for

the last the estimate of the minimal number of copies is 9

from our hybridization studies [14,29]. For a different set of

tRNAs the genes appear to occur in "heteroclusters". It would be

of interest to find out, whether there exists a tRNA gene

organization in yeast comparable to that reported in a cloned

Xenopus DNA fragment [32 ] or in cloned Drosophila DNA fragments

[33].

ACKNOWLEDGEMENTS

We wish to thank Mrs. C. BleifuB for expert technical

assistance, we are grateful for the gift of tRNAs to Drs. G.Dir-

heimer and G. Keith (Strasbourg) and for the gift of restriction

enzymes to Drs. R.E. Streeck and U. Hanggi from this laboratory.

The Deutsche Forschungsgemeinschaft (Forschergruppe Genomorgani-

sation) and the Fonds der Chemischen Industrie have supported

this work.

1984



Judith Olah's present address: Dept. Genet, and cell Biol.Univ. Minn., 250 Biol. Sci. Center, St. Paul, Minn. 55108

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1986


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Structure of a yeast non-initiating methionine-tRNA gene Judith