Current Genetics

Current Genetics (1981) 4:135-143

© Springer-Verlag 1981

Organization and Expression of a tRNA Gene Cluster in Saccharomyces cerevisiae Mitochondrial DNA D. L.Miller and N. C. Martin Department of Biochemistry, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 USA

Summary. We have studied the organization and expression of a group of tRNA genes located on a 2,700 base pair portion of the yeast mitochondrial genome between the genetic markers cap (chioramphenicol resistance) and oxil (cytochrome oxidase subunit II). This region is sFanned by mitochondrial DNA inserts of two recombinant plasmids, pYm162 and pYm267, which have been extensively mapped and sequenced. This tRNA group is composed of six tRNA genes, coding for t R N A ~ R , Arg Gly tRNAAG~y , tRNAAGY, set tRNAAGR, tRNAGGN, and Arg tRNAcGrq. We report the sequence for the majority of the 2,700 base pair region including the genes for all six tRNAs. All six genes are oriented in the same direction and are, therefore, transcribed from the same DNA strand. Further, a comparison of the organization of this region with the analogous region of a related wild type strain shows that the tRNA gene order in the two strains is the same. Five of the six tRNA genes have corresponding transcripts in wild type RNA. Although a potential structural gene for tRNAA~N is present,we do not detect a t R N A ~ N gene transcript. Key words: Saccharomyces cerevisiae - Mitochondria Gene cloning - Transfer RNA

Introduction The yeast mitochondrial genome codes for all of the tRNAs required for mitochondrial protein synthesis Abbreviations: SDS: sodium dodecyl sulfate; EDTA: ethylenediamine tetraacetic acid; tris: tris-(hydroxymethyl) amino meth ane; HEPES: N-2-hydroxy ethyl piperazine-N'-2-ethane sulfonic acid, DMSO: dimethyl sulfoxide; DBM: diazobenzytoxymethyl Offprint requests to: Nancy C. Martin

(Martin et al. 1977;Wesolowski and Fukuhara 1979). 24 tRNA genes have been mapped on the mitochondrial genome and at least one gene corresponding to each of the 20 common amino acids has been found. 16 of the tRNA genes are located within 12% of the genome between the genetic markers C (chloramphenlcol resistance) and o x i l (cytochrome oxidase subunit II) while the others are scattered more widely around the genome (see Fig. 1). We have previously reported the sequences of several tRNA genes from the region of the genome between C and oxil, but the limited sequence and mapping data did not allow a determination of the order and arrangement of the genes with respect to each other and to the restriction enzyme map of the wild type mitochondrial DNA. This earlier work (Miller et al. 1980; Martin et al. 1980a) on two pBR322-mitochondrial DNA recombinants with overlapping mitochondrial inserts has been extended to show that they contain six tRNA genes. We have located the six tRNA genes with respect to restriction sites mapped for the wild type genome and find that the coding sequences are all on the same strand. Three of the tRNA genes located in this group, namely, those coding for the t R N A ~ y , tRNA~Y~ and tRNAAr~ R, have not been reported by us previously and are reported here. Bonitz and Tzagoloff (1980) have also reported a sequence of this region of the wild type genome which they obtained by sequencing a petite mutant containing these six tRNA genes. The sequences reported in that work and the sequences reported here are in substantial agreement differing in only a few bases in A + T spacer regions. This is an expected finding considering that their petite and our recombinants were obtained from the same wild type parent, D273-10B. Extensive deletion mapping of tRNA genes has been accomplished using a different wild type strain, MH41-7B, and the results are summarized by Wesolowski and Fukuhara (1979). Further, Wesolowski et al. (1980) have 0172-8083/81/0004/0135/$01.80

136

D. L. Miller and N. C. Martin: Mitochondrial tRNA Gene Cluster ,

E.C ~

tRNA

O,

Fig. 1. The wild type mitochondrial genome of Saccharomyces cereviseae (after Wesolowski and Fukuhara 1979; Borst and Grivell 1981). E, C, P, and O indicate the antibiotic resistance markers for erythromycin, chloramphenicol, paromomycin, and oligomycin, respectively. The location of genes and various genetic markers are indicated. Dots show the location of the various tRNA genes. Transfer RNA Region I as defined by Morimoto and Rabinowitz (1979) is shown. The bar indicates the location of the cluster of tRNA genes cloned in plasmids pYre162 and pYm267

mapped many t R N A genes relative to various restriction sites b y correlating the restriction maps of petite DNAs derived from MH41-7B w i t h the t R N A genes they have retained or lost. MH41-7B and D273-10B are known to differ in size b y 5,000 base pairs, but this size difference is primarily d u e to insertion/deletions at only a few sites in other portions of the genome (Sanders et al. 1977; Morimoto and Rabinowitz 1979). Until now, the ordering of genes on the two genomes has seemed identical. However, sequence information from the wild type DNA of D273-10B gives a different order of the t R N A genes than that derived b y deletion mapping o f MH41-TB. Since these two strains represent the two major wild type genomes o f Saccharomyces cerevisiae (Sanders et al. 1977), it was o f interest to determine if the differences in gene order were due to the differences in technique or t o real differences between the two genomes. To determine the relationship o f these genomes, we have compared the t R N A genes mapped in strain D273-10B with the order and location of the analogous genes from strain MH41-7B. We find the gene order and structure of this region o f these wild type strains to be similar. Our laboratory (Miller et al. 1979; Miller et al. 1980; Miller et al. 1981 ;Martin et al. 1980a;Martin et al. 1980b; and Newman et al. 1980) and others (Li and Tzagoloff 1979; Bos et al. 1979; Nobrega and Tzagoloff 1980; Bonitz and Tzagoloff 1980; Tzagaloff et al. 1980; Berlani et al. 1980a; and Beflani et al. 1980b) have now se-

quenced nearly all of the known t R N A genes o f the yeast mitochondrial genome. These sequences have yielded information about the structure and transcription o f these genes as well as information about mitochondrial codon utilization. Comparison o f codon utilization in the sequences of mitochondrial genes with the anticodons o f the tRNAs has shown that codon recognition in mitochondrial protein synthesis is different from that ofcytoplasmicprotein synthesis (Bonitz et al. 1980a). Sequence analysis has also shown that some codons are not used in sequences that code for known m i t o c h o n drial protein products. The entire CGN arginine family is composed of such codons, yet a t R N A which would recognize this family could be coded b y a DNA sequence in t h i s region. We find that although the other t R N A genes in this region are transcribed, transcripts from the putative tRNAA~N gene were not detected b y hybridization experiments.

Materials and Methods Yeast and Bacterial Strains. Saccharomyces cerevisiae strains D273-10B (a prototroph) and MH41-TB (a ade2 his1) were obtained from A. Tzagoloff and H. Fukuhara, respectively. All recombinant plasmids were grown and maintained in E. coli strain HB101 (pro- leu- thi- lacY- hsdR- endA- recA- rpsL20 ara14 galK2 yy15 rot1-1 supE44). Construction of plasmids pYm162 and pYre267 was reported previously (Martin et al. 1979). Plasmid pSA14 was constructed by ligation of the HpaII fragment containing the tRNAA~N and tRNASAe~ygenes to the staggered ends of ClaI digested pBR322 using T4 DNA lig~tse (New England Biolabs). To accomplish this, 1-5 pmol of ends of pBR 322 digested with ClaI and treated with phosphatase, and of the HpaII fragment, were incubated together in 50 #1 of 66 mM tris-HC1, pH 7.5, 66 mM MgCI2, 10 mM dithiothreitol, 60 #M ATP and 300 units T4 DNA ligase (P-L Biochemicals, Inc.) at 40 °C for 60 min. After 60 rain the sample was diluted to 250#1 with the above solution and incubated overnight at 14 °C to promote intrastrand ligation. Without further purification, the ligated DNA was used to transform E. coli strain HB101 by the method of Dagert and Ehrlich (1979). Colonies containing the HpaII fragment were selected using a modification of the method of Grunstein and Hogness (1975). Isolation of Nucleic Acids. Isolation of plasmid DNAs was as pre-

viously described (Martin et al. 1979). Isolation of mitoehondrial DNA from isolated mitochondria obtained from zymolyase treated cells using bis-benzimide gradients was as described by Hudspeth et al. (1980). Total mitochondrial RNA was prepared by a single phenol extraction of mitochondrial SDS lysates as described by Locker (1979). Restriction Endonuclease Digests. Restriction enzymes were ob-

tained commercially and the various DNAs digested under the conditions recommended by the commercial source. Radioactive Labeling of Nucleic Acids. DNA fragments with pro-

truding 5' staggered ends produced by restriction enzymes were radiolabeled at the 3' ends of the fragment with E. coli DNA polymerase I (New England Biolabs) and the appropriate [a_32p] deoxytriphosphate. DNA fragments were also radiolabeled at

D. L. Miller and N. C. Martin: Mitochondrial tRNA Gene Cluster

137

p-.~

~--C Ha ADA Hp A

Ha

H Hp

H

T

HaHa

SARH

Ha

Ha T

Ha

I I I-.J

I.--J

I,.,,.J

Lys Arg

L.J

Gly

Asp

4(. . . . . .

I I

pYm

u u

267

nm

~'o. . . . .

u

.1~

4

,no

nu

nnno

I I

ill l

t

n J

SerArg It-

4,

~

I

--

~oz-Vm"~" pt

I

I

I I

I

1Kb

their 5' termini using T4 polynucleotide kinase (New England Biolabs) and calf intestine alkaline phosphatase (BoehringerMannheim). Details of fragment isolation, labeling and gel electrophoresis have been described (Miller et al. 1979). RNA was radiolabeled by ligation of [5'-32p] cytidine 3',5'-Bis (phosphate) to the 3' end of RNA using T4 RNA ligase (P-L Biochemicals). The RNA is radiolabeled by incubation in 50 mM HEPES, pH 7.4, 20 mM MgC12, 6 #M ATP, 11% DMSO, 3.5 mM dithiothreitol, 10/~g/ml bovine serum albumin at 4 °C overnight. Uniform labeling of DNA fragments for use as probes in hybridization experiments was accomplished by nick translation of DNA fragments using the nick translation kit of Bethesda Research Laboratories.

Gel Eleetrophoresis, in situ Transfers and Hybridizations. DNA fragments in agarose gels were transferred in situ to nitrocellulose paper using the method of Southern (1975). RNA fragments were transferred in situ to DBM-cellulose paper by the electroblotting method of Stellwag and Dahlberg (1980). Filters with bound DNA or RNA were incubated with 105106 epm of the appropriate nucleic acid probe in 50% formamide, 750 mM NaC1, 75 mM Na citrate, 50 mM sodium phosphate buffer, pH 6.5, 0.1% SDS, 0.02% fieoI1, 0.02% polyvinylpyrolidone, 0.02% bovine serum albumin and 250 #g/ml calf thymus DNA at 42 °C overnight. The filters were washed two times in 300 mM NaC1, 30 mM sodium citrate, 20 mM sodium phosphate buffer, pH 6.5, and 0.1% SDS; and two times in 15 mM NaC1, 1.5 mM sodium citrate, 1 mM sodium phosphate buffer, pH 6.5, and 0.1% SDS. Autoradiograms were prepared by exposing the washed filters to Kodak XAR X ray film with a Dupont Cronex lightning-plus intensifying screen at -70 °C.

DNA "Sequenee Determination. Sequence determination of the isolated, labeled fragments was obtained by the Maxam and Gilbert (1980) procedttre using the A > C, A + G, G, C and C ÷ T reactions. The sequence between 2 and 50 nucleotides from the labeled ends was obtained by fraetionation of the cleavage products on a 38 cm, 20% polyacrylamide-7 M urea thin (0.5 ram) gel. The sequence from 25 to 250 nucleotides was obtained on an 87 cm 6% polyacrylamide-7 M urea thin (0.5 ram) gel.

Fig. 2. The restriction enzyme map of a portion of tRNA region I containing six tRNA genes. The location of the tRNA genes are indicated by boxes and the three letter code for the cognate amino acids. Abbreviations for restriction enzyme sites are as follows: AluI (A), DdeI (D), HaeIII (Ha), HinfI (H), HpaII (Hp), Rsal (R), San3AI (S) and TaqI (T). The extent of the fragment cloned in the two.recombinant plasmids is indicated below the map. The bar at the bottom indicates the scale in kilobase pairs. C and P indicate the direction of the chloramphenicol resistance and paromomycin resistance markers on-the wild type genome. Arrows below the map indicate the direction and extent of sequence analysis from a labeled end. Dots depict the location of the label. Arrows made of solid lines represent fragments labeled and sequenced from the 3' end; arrows made o f dashed lines represent fragments labeled and sequenced from the 5' end

Results

Restriction Endonuclease Mapping and Sequence Analysis o f the t R N A Gene Cluster The construction of yeast mitochondrial DNA-pBR 322 recombinant molecules and the identification of a subset of such molecules that contain tRNA genes has been reported previously (Martin et al. 1979). We have used these plasmids to deduce the structure o f yeast mitochondrial t R N A genes b y DNA sequence analysis o f several cloned t R N A genes and the spacer DNA immediately adjacent to the coding regions (Miller et al. 1979; Miller et al. 1980; Martin et al. 1980a; Martin et al. 1980b; Newman et al. 1980; Miller et al. 1981), but in the case of some of these genes we had n o t determined the orientation o f the sequenced genes relative to each other or to the mitochondrial genome. In addition, some of the plasmids contained t R N A genes which remained to be sequenced. Two such plasmids, pYm162 and pYm267, are more completely characterized here. These two plasmids contain overlapping inserts which span about 2,700 base pairs of the mitochondrial genome and contain a number of t R N A genes. As demonstrated below, they provide an excellent source of D N A for studies o f the organization and expression of a number o f linked t R N A genes. To determine the relative location o f the t R N A genes, isolated plasmid D N A was digested with a variety o f restriction enzymes and the digested fragments were separated on 2% agarose gels and sized relative to the published sizes o f restriction fragments from pBR322 (Sutcliffe 1978). The results o f single digests and multiple digests of mitochondrial DNA with various combinations of restriction enzymes yielded the physical map shown

138

D.L. Miller and N. C. Martin: Mito chondrial tRNA Gene Cluster

L~/s

O00IccGd . . . . . . . . . . . . Hpa I I

GC6C CCAAAGdAGTAATATATATTATGTATAAAC AATAC, A6t~\T ATT(.';TTTAAT d(:.Tzk&,b\(:;,\£; TT(iT(':TTTTA

Ali[:A..\('X~(~AT(31 C~O

010IGCTTdGTT6A ACTCCAdCTA TTCTCATAAT ATTATATATA TATATTTCCC TTTCTAAAAA TAATAATAATTATATATAAT ,V~T,b~,T,\T;u\ TTATATATAT02I]0 Alu I .010o 0201ATATATTATA ATAATAATAA TAATAATAAT TAATAATAAT AATTATTTTT ATTAATAATA TTAATATATT AT*~\TTATTA ATAAATATTAA T ~ T A C Alu [

Arg

0301CTCTCTTAGC TTAATGGTTA AAGCATAATA CTTCTAATAT TAATATTCCA

TdTTCAAATC ATC,C,AC,AC,AC, TA~\TTATATT ATATTAAT,kAT('.'00(:X':('X)('b\n400

Dde I Alu I

Gly.

o~oo

0401TTTTTAATTT TATTAAGAAGTTTAATTTAC TATTTAATAATAAATGAAAT AATAATAATA GATATAAGTTAAITCGTAAA CTC,C,AT(';TCT TCCAAACATT 0501;AATGCGAGT TCGATTCTCG CTATCTATAA TTAATATTAA TATAAATTAA Taq I Hinf I

TATCCTATAA TTAATTAAATACAAAATTATATTAAAACTT ATATTATATTnhOn

0601ATAATATTAT ATTATTATTA T A T A A ~ T A

TTTATTTAAT AATAATATTTTATATAATAAAATAATCATATTTATAATAT070~

0701TTAATATTAA

TAATAATAAT AATATTTAAT

.............. about 245 nueleotides .......

i001 .......................

~66 ....................

AAA

. . . . . CCGG. . . . . . . . . . Hpa II

about 70 n u c l e o t i d e s . . . . . . . . . . . .

lOo0

AAGTATATAT AATATAATTA ATATATTTCT TTTTATATAA ATTATAAATA llO0

Hpa II

II01TTAATTTATA ATAAAAAAAG TATATATAAT ATTATATATT TAATAAATAA

TATAATAATA ATATAAATAA ATATATATAT ATTATTAATA TATTAAATTT

1201TATAATAATA ATTATAATAA TAGTAGTAGG TATAAATTTT AATAAAGAGT

TTTATTCCAA TGdA6TAATA ATAATAATAA TAATAAAATA AAG6ATCTGT Sau3A I

1301AGCTTAATAG TAAAGTACCA TTTTGTCATA ATGGAGGATG TCAGTGCAAA Alu I Rsa I 1401ATATATATAT TTAATAATAT TTTTCTTTAT TATAATAATA TATAAAAATA

1501TTATTATTAT TAATTTATAT TAATATTTTA TATAAATTAT

Asp.. t3o0

. . . . . . 1400 TCTGATTAGA TTCGTATATT AATACTT&AT ATAAAAAAAT AAATAATAAA Hinf I .1500 AATAATAATC TTTTTTTTAT TATTATATTT ATTAATAATA AATTATTTTC

...................

... ........ 1701 ...................... CCGGGCGG GGACTTATTT TTATATTTAT Hpa II

1200

about 185 nucleotides . . . . . . . . . . . . . . . . . . . . . .

1700

1800 TAATAATAAT TAATTTATTA TTTCTTACAA TATATTTATT ACTATTATTT

1801TTTAATAAT¢ TTATATATAA TATATAAAAT ATATATATAT TATATATATA

TATAAATATA ATATATATTA TTATAAATAT TrATAATCTT ATTAATTAAT 1900

1901TAGATTATAT TATATTATAT TAGATCATAT TATATTATAT TATATTATAT Sau3A I

TATATTATTA TTATTAATAT TTTTATTTTT ATTTTATATT TAATAGTAAA 2000

2001AJ~ATCATAAT

....... TTT TATTTATTTA TTTATTACTT ATTAATAGTT CCGG ...... 2100 Hpa II

2101

TTTATAATTT ATTAATTATT ATATAATTTC A .........

..............................

AAAAAATAAT TATAATTTAT

TATAATTTAT TAATTTATTA ATTTATTAAT TTATTTATTA ATTTATTAAT 2200

Set

2201TTATTTATTA TTATATTTTT TTTAATAAAG GAAAATTAAC TATAGGTAAA

Arg 2301TCATATT~TC CGTATATA__~A.T~TTTAATTTAA TGGTAAAATA TTAGAATACG

GT;;ATTATT T;CTAA;TAA TT;AATT;TA AATTCTTAT; A;TTCGAATC2300 Taql Hlnfl AATCTAATTA TATAGGTTCA AATCCTATAA GATATTATAT TATATTATAT 2400

HlnfI

2401AATATTATAT ATTAATAAAT ATTATTAATT AATTTATTTA TTTATTTATT 2501

ATTAAATAAA AATATTTAAT AGTTCC ................. CCGG... 2500 Hpa II

.................. TA TTATAATTAT TTATATATTA ATTATTAATT

ATTTATTATT TATTATATAA AAAGTATATA ATTTTATATT TTAATATAGG 2600

2601GTTAATTAAT TAATTATTAA TTTTTTATAA TAAGXTAATA ATATATTAAA

AACTTATTAT AAATTTATAA AATAATAI-fT ATTTACTTTG ATATTATTTT 2700

2701TAATTC

Fig. 3. The DNA sequence of a portion of the noncoding strand of the 2,700 base pair region containing the tRNA gene cluster. The portion of the sequence correspondi, g to the tRNA sequences is underlined• Restriction endonuclease cleavage sites are shown below the sequence. Dots have been placed above the Gs and Cs to emphasize their distribution

in Fig. 2. Hybridization o f the various restriction enzyme fragments with mitochondrial t R N A gave the general location o f several tRNA genes on the physical map o f the mitochondrial DNA. In order to determine which tRNA genes were encoded within the mitochondrial DNA and the orientation o f these genes, we have sequenced the

majority of the mitochondrial D N A from the two plasraids. This sequence is shown in Fig. 3. The direction and extent o f sequence analysis is shown b y arrows in Fig. 2. When this DNA sequence is searched for sequences which could code for tRNAs, six such regions are found. They correlate well with the locations o f t R N A genes de-

D. L. Miller and N. C. Martin: Mitochondrial t R N A Gene Cluster A

A

Lys

A

pG -- c

pG - U C-G u --A C-G U--A C-G

A-U G-C A--U A--U U--A

A--U

o'"

o°

CA

~,?;~° I I

A

GuAA

u G C U u GG U U c

A AG_cCC

u-% IIII

G

AAGC UUA

A

i

Gly

GCGAGuuC U A

I I

AAAC u

The overall order and arrangement of the tRNA genes in strain D273-10B as shown in Fig. 2 is different from the order of tRNA genes predicted by deletion mapping from strain MH41-7B (Martin et al. 1977; Wesolowski and Fukuhara 1979). Since the reported location of these genes on restriction fragments in MH41-7B (Wesolowski et al. 1980) depends on the deletion mapping, it is also different from that obtained from sequence analysis of D273-10B. Taken together, the previously published studies of MH41-TB locate the tRNAA~y gene beyond the tRNALA~R gene in the direction of C, and locate the t R N A ~ N gene beyond the tRNAAG¥ set gene in the direction of the oxil locus. They show that the tRNAA~y, tRNA2~SR , t R N A ~ R , and tRNAS~y genes are clustered within a 1,020 base pair region which is contained within two MboI fragments of 6,000 and 630 base pairs (MboI and Sau3A I are isoschisomers) or within two Hinfl fragments one of which is 900 base pairs. They also preLys dict that th e tRNAGG N gene is on a different HinfI arid MboI fragment than the tRNA~Y~ and tRNAA~R genes. To determine whether these two wild type strains differ in their tRNA gene order and arrangement in region I, as predicted from the above mentioned studies, several small radiolabeled fragments containing specific tRNA genes were prepared. The size and location of the fragments are shown in Fig. 5. These probes were each hybridized to MboI and HinfI digests of both wild type strains. The results of these hybridizations are shown in Fig. 6. In each case the hybridization pattern for the two strains is essentially the same indicating that the tRNA order and arrangement in the two strains gene are similar if not identical. Further, in contrast to the predictions for

pG - G G-U A-U U-A C-G U-A uG-UuAGucUAA

UA

I I I I I

AUUGA

The tRNA Gene Order and Arrangement of Two Different Wild Type Strains

~~' A s p

A pA-U U--A A-U G-C A-U U-A AU A - U C G C U C U

U

AA A

uuC

Uu C u A

UuU

GG

UA AU --A AU

o

A-U U--A cA_UA

g

I I

CCAUG

U

A--U

U-A G-C U-G

u A

....

i I i I I

UUCGA

U--A

U

al. 1980). Additional cloverleaf structures for tRNAk~R, tRNAAr~R, t R N A ~ N , and tRNAA~y are in Fig. 4. The sequence of the tRNAG~N varies from that which we reported previously (Miller et al. 1980) and we wish to amend the initial sequence to read as shown in Fig. 3 and 4.

~ A rg

A

UUUO G I I I I GuAAAA C

139

G

AA A I I I I i A O UUCG GUCAG C A I I I I UA UG GUAAAG o A C _ G AGG C-G A-U U--A U-A

GG_uG A -U U-A G-C U-A C A

GuC

UcC

Fig. 4. Cloverleaf representations o f t R N A s deduced from their DNA sequence. Transfer RNAs for aspartic acid, glycine, lysine and arginine are shown. The 3' CCA sequence which is n o t encoded b y t h e DNA has been added. The locations of modified bases are n o t k n o w n

termined from the hybridization of mitochondrial tRNA with restriction enzyme digests of these plasmids. Transfer RNA structures deduced fromthese six sequences indicate that they code for t R N A ~ R , tRNAAar~R,t R N A ~ N , Asp tRNAA SerGY, and tRNA~r~N. All six sequences tRNAGAy, are arranged in the same orientation and would, therefore, be transcribed from the same DNA strand. The 945 base pair HpaII fragment from plasmid pYm162 contains the sequences for threetRNAs. They are Gly N , tRNAA~GGg,and t R N A ~ R. The gene for tRNAGG t R N A ~ R is separated from the tRNAcG GlyN gene by 85 base pairs. The gene for t R N A ~ R is located 173 base pairs distal from the tRNAA~R sequence. We have reported the sequences of the tRNAs deduced from their ArgN (Martin et DNA sequence for tRNASe~y and tRNAcc

1

5 6 I'--I I'---I 4 I' I

2

-~"C Ha H Hp

Ha

I I

.,n,, Sau3A

I I

ADA

J--J J--J Lys Arg

1100

H

YT

1,r°S~iAhtl¢. .

L~I Gly

I 6000*

HaHa

Ha

l~p ~

L.J Asp

6.6

I ..o

]630 t

Ha Hp l

.H'.DTHIH ' J • SerArg

5.+1

p--~

;

3.oo 5700*

Fig. 5. Location of the labeled probes used to determine t R N A organization on t h e restriction m a p of t h e t R N A cluster. Bars denote the location and e x t e n t of t h e individual probes. The probes are numbered for reference. Restriction e n z y m e sites are defined in Fig. 2. Boxes represent the location of t R N A genes. The size and location of t h e restriction fragments for Hinfl and MboI from this region are shown below the map. Those sizes marked with an asterisk are from Wesolowski et al. (1980)

140

D . L . Miller and N. C. Martin: Mitochondrial t R N A Gene Cluster

Fig. 6 A and B. Autoradiograms o f hybridizations o f various probes with MboI (A) and Hinfl (B) digests of yeast mitochondrial DNA from strains MH41-7B and D273-10B transferred to nitrocellulose paper. The letters M and D refer to the strains MH41-TB and D273-10B respectively. The letter E designates the ethidium bromide stained gel containing the digested DNA fragments. The n u m b e r s 1, 2, 3, and 4, refer to the probes used in t h e hybridization as defined in Fig. 5. The n u m b e r s at the side are the size of the DNA fragments in base pairs

MH41-7B, the tRNAA~7, tRNAL~R, tRNAcGN, Arg and tRNASe~y genes hybridize to three different MboI frag- --Gly ments; and tRNAGG N gene is on the same HinfI and MboI fragment as the tRNAL~R and t R N A ~ R genes. Aside from the few differences mentioned above, the corrected and extended deletion map of MH41-7B tRNA genes (Wesolowski and Fukuhara 1979) and the correlation with the MH41-7B restriction map (Wesolowski et al. 1980) is in good agreement with the results of sequencing studies. Comparison of the gene cluster region with the MH41-TB map allows one to determine the orientation of the plasmid mitochondrial DNA on the wild type genome. This indicates that the gene cluster is transcribed in the same direction as all the other mitoThr chondrial genes except the tRNAcu N gene (Li and Tzagaloff 1979).

Transcription of tRNA Genes To analyse the transcripts of these genes, RNA from isolated yeast mitochondria was separated on agaroseurea gels, electroblotted to DBM-cellulose paper and hybridized with various tRNA-specific probes. The tRNAALySR -tRNAA~R probe 1 and the t R N A ~ p probe 3 (see Fig. 5) both hybridize with 4S transcripts which are presumably the mature tRNAs coded by these genes (data not shown). Even at very long exposure times no hybridization was seen to higher molecular weight RNAs. Ser The fact that the sequences for tRNA2G Y and tRNAA~N are separated by only three base pairs suggests that these two genes might be initially transcribed as at least a dimeric precursor and subsequently proces-

sed to the mature transcript. To obtain a large quantity of purified probe, the HpaII fragment containing these two sequences (Fragment 4, Fig. 5) was cloned in the ClaI site ofpBR322. From the resultant plasmid, pSA14, two probes were prepared. One probe similar to probe 5 (Fig. 5) contained only tile tRNA2c set Y sequences and one probe similar to probe 6 (Fig. 5) contained only t R N A ~ N sequences. These probes were separately hybridized with filters containing RNA transferred in situ from denaturing gels of mitochondrial RNA. Easily detectable amounts of tRNA~,Gy set gene transcripts are present in mitochondrial RNA, but no detectable hybridization could be observed with the tRNAA~N specific probe (Fig. 7A). This result was confirmed by the reciprocal experiment. When total 32p-mitochondrial tRNA was hybridized with HinfI digested pSA14, only the 1,220 base pair Hinfl fragment containing the Ser tRNA~G ¥ gene hybridized tRNA (Fig. 7B). The 750 base pair Hinfl fragment containing the tRNAcG argN gene did not.

Discussion

We have studied two recombinant plasmids, pYm162 and pYm267, which contain yeast mitochondrial DNA cloned at the EcoRI site of pBR322 (Martin et al. 1979). These plasmids contain overlapping segments of tRNA region 1 as defined by Morimoto and Rabinowitz (1978). We have mapped and extensively sequenced the mitochondrial portion of both plasmids. Hybridization of total mitochondrial tRNA with various restriction digests of the plasmids transferred in situ to nitrocellulose paper have shown that there are at least six different genes, of

D. L. Miller and N. C. Martin: Mitochondrial tRNA Gene Cluster

Fig. 7 A and B. Hybridization of mitochondrial RNA with DNA fragments specific for tRNASAe~yand tRNAcA~gN. Panel A: Mitochondrial RNA was electrophoresed in 1.5% agarose-6Murea gels, stained with ethidum bromide (E) and transferred in situ to DBM paper. The numbers 5 and 6 indicate the autoradiograph of the DBM filter following hybridization with the tRNAS~y specific probe (number 5, Fig. 5), and tRNA~gN specific probe (number 6, Fig. 5) respectively. Panel B: Hinfl pSA14 DNA fragments were electrophoresed in a 2% agarose gel, stained with ethidum bromide (E) and transferred in situ to nitrocellulose paper for hybridization with radiolabeled mitochondrial RNA (R). The numbers at the side refer to the size of the DNA fragments in base pairs. Set and Arg indicate the mitochondrial fragments which contain the tRNAA~Y and tRNA~,q genes respectively

which at least five are transcribed, located within about 2,700 base pairs. While these coding regions are clustered within the genome, there is still considerable separation of the genes. The exceptions to this rule are the tRNASAe~y and t R N A ~ N coding regions which are located only three base pairs apart. Otherwise, the tRNA sequences are separated by relatively large regions of very high A + T content. Six G + C rich site clusters (Prunell and Bernardi 1977) are also present in this 2,700 base pair sequence. PruneU and Bernardi (1977) have proposed that these G + C rich sequences could be promotors, while Tzagoloft et al. (1980) propose that they may provide secondary structure needed for tRNA processing. However, the location of the site clusters bears no obvious relationship to the genes themselves. While three genes are located between a single pair of site clusters in one case, several site cluster pairs do not contain any genes between them. The most striking thing about the arrangement of the tRNA genes is that they are all in the same orientation and could, therefore, be transcribed together from the same strand. This is the same strand from which nearly all of the other mitochondrial genes are transcribed. This gene arrangement is consistent with a model in which

141 the entire coding strand in wild type cells is transcribed and the mature RNAs produced by post-transcriptional processing. However, if this occurs it must be very rapid in vivo because no higher molecular weight RNAs containing tRNA sequences from these genes were detected. The sequence of the genes for t R N A ~ R , tRNAcay,ASp and tRNALA~R (Fig. 3.) are typical of mitochondrial tRNA genes in that they do not contain intervening sequences nor do they code for 3' CCA termini. The tRNAA~R predicted from the DNA sequence has the typical structure and conserved bases of classical tRNAs. The tRNAA~y is unusual in that it has a single G in the D loop and a - U G C A - instead of the more common -UUGC (T~GC) in the Tt~ loop. The tRNAL~R has the unusual feature of an extra, unpaired uridine in the pseudouridine stem (see Fig. 4). This feature has also been observed in the tRNAuuv Phe from yeast mitochondria (Miller et al. 1979; Martin et al. 1979)and is otherwise unprecedented. These two genes do not have extensive homology. It is not clear if this structural feature has a function. Different strains ofSaccharomyces cerevisiae have differently sized mitochondrial DNA. Sanders et al. (1977) and Morimoto and Rabinowitz (1979) have mapped the two major types of mitochondrial DNA with different restriction enzymes that each cut at only a few sites on mtDNA, and have found that the larger genome (75 kb) is similar to the smaller (70 kb) genome but has some extra sequences at a small number of sites. Prunell et al. (1977) have compared digests of mitochondrial DNA from several different strains with restriction enzymes that cut at many sites on the mitochondrial DNA. They find that the location of many of the these sites varies between strains. So,in addition to the major addition/deletion differences, a microheterogeneity between strains exists. It was of interest to determine if this heterogeneity affected the arrangement and order of the tRNA genes on the different genomes. The fact that deletion mapping of MH41-7B and sequence analysis of D273-10B gave different gene orders suggested that this might be possible. Restriction digests coupled with hybridization of specific tRNA gene probes strongly suggests the order of tRNA genes in this region is the same in both wild type strains. This argues for the essential similarity of these two strains and lends validity to the extrapolation of results from one strain to the other. We have also analysed the transcripts of the tRNA genes in this region. DNA probes for tRNA~A¥ Asp , tRNAk~R, tRNAAA~R and ÷DxIASer . . . . ~AGY hybridize only with 4S RNA. This is not surprising since aminoacyl tRNAs corresponding to tRNAG~N, tRNAGAY, " Asp f~.l .~. a. .SAe rGY, tRNALY~, and to one isoacceptor of tRNA Arg are known to hybridize to this region of the genome, and therefore, at least one tRNA gene corresponding to each: of these tRNAs must be expressed. The tRNpaAr~ is

142

D.L. Miller and N. C. Martin: Mitochondrial tRNA Gene Cluster

necessary for mitochondrial protein synthesis (Bonitz et al. 1980a) and is expressed. Expression o f the tRNA~G~N is more open to question. While the sequences o f the tRNASe~y gene hybridize strongly with 4S RNA, the t R N A Arg c 6 N gene sequence only three base pairs away hybridizes little if at all with mitochondrial RNA. Since the codons decoded by this tRNA~gN are not used in any known mitochondrially encoded proteins (Bonitz et al. 1980a), one might suppose that mitochondrial protein synthesis could continue without this tRNA. On the other hand, there is growing evidence that open reading frames present within certain introns of mitochondrial proteins are translated in catalytic amounts. These seArgN codons and their translaquences contain tRNAcG tion would require at least small amounts of t R N A c ~ N to be produced (Nobrega and Tzagoloff 1980; Lazowska et al. 1980; Bonitz et al. 1980b). Even though we cannot detect such a tRNA by hybridization the possibility that it is produced in small amounts cannot be ruled out. Production of functional tRNAA~N would be o f interest for several reasons. First, the tRNA sequence corresponding to the gene for tRNAA~N is unique in that it is the only mitochondrial tRNA with an adenosine residue at the wobble position. Adenosine has never been found in this position in any tRNA (Gauss et al. 1981), but is invariably modified to an inosine residue. If the same rule holds for mitochondrial tRNAs, then the tRNAA~N would contain inosine at the wobble position. That Martin et ai. (1976) have detected a small amount o f inosine in isolated mitochondrial tRNA might indicate that the ArgN sequence is expressed. However, these low tRNAcG amounts of inosine could also be due to small amounts of contaminating cytoplasmic tRNAs. Second, the mechanism by which a gene can be expressed while a similar sequence three base pairs away is not expressed is unclear. If both the tRNAAG Ser v and the t R N A c ~ ~ genes are expressed, then either the transcription rate of each gene is radically different or the stability of their transcripts varies widely. Differential rates of transcription could occur if there are separate promotors for eachgene. If promotors are outside of, but close to the coding region then the promotor for the t R N A ~ N gene would have to be in the t R N A ~ y gene. Since nothing is known about mitochondrial promotors, this is possible. It is also possible that sites internal to the genes control polymerase binding as has been found in the 5S RNA genes of Xenopus (Sakonju et al. 1980; Bogenhagen et al. 1980; Engelke et al. 1980). Yet another way that the *W'TA set and t R N A c ~ N genes v ~ AGY could be differentiany transcribed is if both were promoted from the same site but transcription was most often terminated after the tRNAAS~y gene. I f both genes are transcribed together in the same transcript, differential stability of the transcripts could explain why t R N A ~ y --Arg is easily detected and tRNAcG N is not. Clearly, further

studies on the possible transcription and role of tRNAA~N in mitochondrial protein synthesis are necessary.

-

Acknowledgements. This work was supported by grants to NCM from the National Institutes of Health and the Robert A. Welch Foundation.

References 1. Berlani R, Pentalla C, Macino G, Tzagaloff A (1980a) J Bacterial 141:1086-1097 2. Berlani RE, Bonitz SG, Coruzzi G, Nobrega M, Tzagoloff A (1980b) Nucleic Acids Res 8:5017-5031 3. Boge.nhagen DF, Sakonju S, Brown DD (1980) Cell 19: 27-35 4. Bonitz SG, Berlani R, Coruzzi G, Li M, Macino G, Nobrega FG, Nobrega MP, Thalenfeld BE, Tzagoloff A (1980a) Proc Natl Acad Sci USA 77:3167-3170 5. Bonitz SG, Coruzzi G, Thalenfeld BE, Tzagoloff A, Macino G (1980b) J Biol Chem 255:11927-11941 6. Bonitz SG, TzagaloffA (1980) J Biol Chem 255:9076-9081 7. Borst P, Grivell LA (1981) Nature 290:443-444 8. Bos JL, Osingia KA, Vanderhorst G, Borst P (1979) Nucleic Acids Res 6:3255-3266 9. Dagert M, Ehrlich SD (1979) Gene 6:23-28 10. Engelke DR, Ng S, Shastry BS, Roeder RG (1980) Cell 19: 717-728 11. Gauss DH, Bruter F, Sprinzl M (1981) Nucleic Acids Res 9:rl-r23 12. Grunstein M, Hogness DS (1975) Proc Natl Acad Sci USA 72:3961-3965 13. Hudspeth M, Shumard D, Tatti K, Grossman L (1980)Biophys Biochem Acta 610:221-228 14. Maxam AM, Gilbert W (1980) Sequencing End4abeled DNA with Base-Specific Chemical cleavages. In: Grossman L, Moldave K (eds) Methods in Enzymology, Nucleic Acids Vol 65, Part I. Academic Press, New York, pp 499-560 15. Lazowska J, Jacq C, Slonimski P (1980) Cell 22:333-348 16. Li M, Tzagaloff A (1979) Cell 18:47-53 17. Locker J (1979) Anal Biochem 98:358-367 18. Martin NC, Rabinowitz M, Fukuhara H (1977) Biochemistry 16:4672-4677 19. Martin NC, Miller DL, Donelson JE (1979) J Biol Chem 254:11729--11734 20. Martin NC, Miller D, Hartley J, Moynihan P, Donelson J (1980a) Cell 19:339-343 21. Martin NC, Pham HD, Underbrink-Lyon K, Miller DL, Donelson JD (1980b) Nature 285:579-581 22. Martin R, Schneller JM, Stahl AJC, Dirheimer G (1976) Biochem Biophys Res Commun 70:997-1002 23. Miller DL, Martin NC, Pham HD, Donelson J (1979) J Biol Chem 254:11735-11740 24. Miller DL, Sigurdson C, Martin NC, Donelson JE (1980) Nucleic Acids Res 8:1435-1442 25. Morimoto R, Rabinowitz M (1979) Mol Gen Genet 170: 25 -48 26. Newman D, Pham HD, Underbrink-Lyon K, Martin NC (1980) Nucleic Acids Res 8:5007-5016 27. Nobrega FG, Tzagoloff A (1980a) FEBS Letters 113:52-54 28. Nobrega FG, Tzagoloff A (1980b) J Biol Chem 255:98219827 29. Purnell A, Bernardi C (1977) J Mol Bio1110:53-54

D. L. Miller and N. C. Martin: Mitochondrial tRNA Gene Cluster

143

30. Purnell A, Kopecka H, Strauss F, Bernardi G (1977) J Mol Biol 110:17-52 31. Sakonju S, Bogenhagen DF, Brown DD (1980) Cell 19:1325 32. Sanders JPM, Heyting C, Verbeet MP, Meijlink FCPW, Borst P (1977) Mol Gen Genet 157:239-261 33. Southern EM (1975) J Mol Biol 98:503-517 34. Stellwag EJ, Dahlberg A (1980) Nuc Acid Res 8:229-318 35. Sutclfffe JG (1978) Cold Spring Harbor Symp 43:77-90 36. Tzagoloff A, Nobrega M, Akai A, Macino G (1980) Curt Genet 2:149-157

37. Wesolowski M, Fukuhara H (1979) Mol Gen Genet 170: 261-275 38. Wesolowsld M, Monnerot M, Fukuhara H (1980) Curr Genet 2:121-130

Note Added in Proof

Dr, R. P. Martin (personal communication)has recently found that the yeast mitoehondrial tRNAcA~gNgene codes for a functional tRNAcA~]q. This tRNA is a minor species, representing approximately 0.1% of the total mitochondrial tRNA.

Communicated b y C.W. Birky, Jr. Received May 11, 1981