Genes, variant genes and pseudogenes of the human tRNAVal gene family expression and pre-tRNA maturation in vitro

J. Mol. Biol. (1989) 209, 505-583

Genes, Variant Genes and Pseudogenes of the Human tRNAVal Gene Family Expression

and Pre-tRNA

Maturation

in Vitro

Hans-Ulrich Thornann?, Cornelia Schmutzlerf-, Uwe Hiidepohl Margret Blow and Hans J. Gross Institut fiir Biochemie Julius-MaximilinnsUniversittit R6ntgenring 11 D-8700 W~iirzburg Federal Republic of Germany

Bayer&he

(Received

4 April

1989)

Nine different members of the human tRNA”“’ gene family have been cloned and characterized. Only four of the genes code for one of the known tRNA”“’ isoacceptors. The remaining five genes carry mutations, which in two cases even affect the normal threedimensional tRNA structure. Each of the genes is transcribed by polymerase III in a HeLa cell nuclear extract, but their transcription efficiencies differ by up to an order of magnitude. Conserved sequences immediately flanking the structural genes that could serve as extragenic control elements were not detected. However, short sequences in the 5’ flanking region of two genes show striking similarity with sequences upstream from two Drosophila melanogaster tRNA”“’ genes. Each of the human tRNA”“’ genes has multiple, i.e. two t,o four, transcription initiation sites. In most cases, transcription termination is caused by oligo(T) sequences downstream from the structural genes. However, the signal sequences ATCTT and CTTCTT also serve as effective polymerase III transcription terminators. The precursors derived from the four tRNAVa’ genes coding for known isoacceptors and those derived from two mutant genes are processed first at their 3’ and subsequently at their 5’ ends to yield mature tRNAs. The precursor derived from a third mutant gene is incompletely maturated at its 3’ end, presumably as a consequence of base-pairing between 5’ and 3’ flanking sequences. Finally, precursors encoded by the genes that carry mutations affecting the tRNA tertiary structure are completely resist’ant to 5’ and 3’ processing.

1. Introduction

tRNA represent large clusters of tRNA genes that are irregularily distributed and may be transcribed from either direction. In rat and in X. Eaevis. a tenfold and a loo-fold repeated unit, respectively. each coding for several tRNA genes, have been reported. Human tRNA genes either occur as isolated entities or as small clusters, which seem to be organized like subcompartments of the large D. melanogaster tRNA gene clusters. It is not known how these organization patterns affect the expression of these genes. Although polymerase III promoters are intragenie, flanking sequences also have a marked influence on the transcriptional properties of tRNA genes (for a review, see Geiduschek & TocchiniValentini, 1988). Yeast and D. melanogaster, respectively, have some common features of extragenie tRNA gene transcription regulation

A growing body of information is accumulating concerning the multigene families coding for tRNAs in higher eukaryotes. Studies have been reported involving the tRNA genes of Drosophila melanogaster (Addison et al., 1982; Leung et al., 1984; Cribbs et al., 1987), Xenopus laevis (Miiller & Clarkson, 1980)) rodents (Sekiya et al., 1982; Makowski et al., 1983; Rosen et al., 1984; Shibuya et al., 1985; Morry & Harding, 1986), and humans (Ma et al., 1984; Shortridge et al., 1985; Pirtle et al., 1986; Arnold et al., 1986; Doran et al., 1987, 1988). In D. melanogaster, more than 50 different sites on polytene chromosomes hybridizing against total

the

t The work reported here was contributed equally by first 2 authors.

OO~‘L~~X3S/SQ/ZoO,5O.5-19

$03.00/O

505

0

1989 Academic

Press

Limited

506

H.-U. Thomann et al.

(Raymond & Johnson, 1987; Sajjadi & Spiegelman, 1987), but there is still no comparable evidence for mammalian tRNA genes. In vertebrates, only a few major tRNA isoacceptor species are found (Sprinzl et al., 1989), whereas much more sequence variations are detected on the tRNA gene level. The variations can comprise the exchange of a few nucleotides, the respective genes have been termed allogenes (Leung et al., 1984), as well as heavily altered or even truncated pseudogenes. There is no information concerning whether all these members of a tRNA gene family are active in vivo or whether there is a yet unknown function for the variant genes or even for the pseudogenes. Transcription of eukaryotic tRNA genes initiates some nucleotides upstream (Galli et al., 1981; Hipskind & Clarkson, 1983) and terminates, in general, at four or more consecutive thymidine bases downstream from the structural genes (DeRobertis & Olson, 1979; Garber & Gage, 1979; Hagenbiichle et al., 1979; Koski & Clarkson, 1982; Schaack et al., 1984; Frendewey et al., 1985). Therefore, tRNAs are first synthesized as precursors that contain extended 5’ leaders and 3’ trailers and, in some cases, intervening sequences. Processing reactions in the nuclear compartment are necessary to yield mature tRNAs. These include removal of additional 5’ and 3’ sequences, splicing, addition of a 3’-terminal CCA sequence and modification of some bases (for reviews, see Clarkson, 1983; Sharp et al., 1985). Newly synthesized pre-tRNAs are not able to enter the cytoplasm by simple diffusion. Transport through the nuclear membrane is obviously coupled with 5’ and 3’ end processing (Melton & Cortese, 1979; Melton et aZ., 1980; Zasloff et aE., 1982a,b; Zasloff, 1983). It has been observed that certain tRNA genes are correctly transcribed, but that the resulting pretRNAs are not processed to functional tRNAs (Harada et aZ., 1984). Ganguly et al. (1988) reported that an intron-containing suppressor pre-tRNAL”” was correctly processed and spliced in vitro, whereas in vivo only splicing occurred. These findings led to speculations as to whether tRNA precursors, especially those stable in vivo, have a yet unknown function. Pre-tRNAs carrying mutations that affect either their overall shape and/or conserved bases are usually not processed in vivo or in vitro (Nishikura et al., 1982; Zasloff et al., 1982c; Harada et al., 1984; Traboni et al., 1984; Pearson et al., 1985; Tobian et al., 1985; Willis et al., 1986; Kahnt et al., 1989). Maturation enzymes are obviously able to distinguish between pre-tRNAs with a correct tRNA domain structure and those with an incorrect structure (Zasloff et al., 1982a). Here, we present the sequences of five newly cloned human tRNA”“’ genes and their flanking sequences, part of which contain Alu repeated elements. Together with four known tRNA”*’ genes (Arnold et al., 1986; Kahnt et al., 1989), we now have available the sequences of nine different members representing more than half of the gene

family estimated to contain at least I3 gene copies (Arnold et al., 1986). Only four of these genes code for the known isoaccepting tRNA”“* species, while the remaining five carry mutations in their structural genes. However, all genes are transcribed in a HeLa cell-free extract. In order to distinguish between putative pseudogenes and presumably functional tRNA genes, we determined the structures of the resulting precursors and the sequence of the processing events. Six of the pre-tRNAs are processed correctly to mature tRNAsVa’. Two precursors derived from mutant genes have altered tertiary structures, which renders them resistant to 5’ and 3’ processing. A third pre-tRNA is not fully maturated at its 3’ end, whereas normal 5’ end maturation occurs. 2. Materials

and Methods

(a) Enzymes and chemicals RNase A and enzymes for molecular cloning were purchased from Boehringer-Mannheim. RNase T, was obtained from Sankyo/Calbiochem. AMV reverse transcriptase was a gift from Dr Horst Domdey, Miinchen. [c+~*P]UTP and [c+~*P]GTP with a specific activity of 15x lOi* Bg per mmol and DJ-~*P]ATP with a specific activity of 110 x 10 ‘* Bg per mmol were from AmershamBuchler. Cellulose acetate strips (55 cm x 3 cm) and DEAE thin-layer plates (20 cm x 40 cm) were from Macherey-Nagel, Diiren, Germany. All other chemicals were from Merck, Darmstadt or Serva, Heidelberg. X-ray films were from Fuji, Tokio. A 15mer oligonucleotide was synthesized using the DNA-synthesizer 2000 from Applied Biosystems. (b) Cloning

and screening procedures

High molecular weight DNA from human placenta was prepared by the method of Blin & Stafford (1976). digested with EcoRI, electrophoresed on 08% (w/v) agarose gels and fractions containing restriction fragments with the molecular weight of interest, i.e. from 4 to 7.5 kbt (population l), from 7.5 to 12 kb (population 2), and longer than 12 kb (population 3), were isolated separately by electroelution. The fragments were ligated into lambda gtWES/lambda B (fragments from 4 to 10 kb) or into lambda. EMBL4 (fragments longer than 10 kb). Hosts for both phages were Exherichia coti BHB2600 and LE392, respectively. Preparation of packaging extracts from lambda lysogens BHB2688 and BHB2690 and packaging of recombinant lambda DNA in vitro were carried out according to standard methods (Hohn, 1982). Screening of recombinant phages was done as described by Woo (1979) with 3’ or 5’ end-labelled rabbit liver tRNAVa’ (spec. act. 1.7 x 10’ to 68 x lo5 cts/s per pg) as a hybridization probe that has 100% identity with human tRNAVa’ (Chen & Roe, 1977; Jank et al., 19775). Positive plaques were purified, and phage DNA was isolated as described by Maniatis et al. (1982) and analysed by standard methods (Southern, 1975). pHtV5 was obtained by screening a human genomic library in phage lambda Charon 4A (Lawn et al., 1978). The library was a gift from Dr T. Maniatis, Cambridge (U.S.A.). t Abbreviations used: kb, 10’ bases or base-pairs; tDNA, tRNA gene: bp, base-pair(s).

A Human tRNA Gene Family Recombinant DNA work was performed with permission and in accordance with the rules of the Zentrale Kommission fiir die Biologische Sicherheit of the Robert Koch-Institut of the Bundesgesundheitsamt. (c)

DNA

sequencing

and

sequence

comparisons

DNA sequences were determined by the dideoxy chain termination method of Sanger et al. (1977) using singlestranded and double-stranded templates. Subclones for sequencing were generated in Ml3 mp18, Ml3 mp19 or in pUC19 either by shotgun techniques or by the systematic E~oIII/E~oVII procedure described by Yanish-Perron et al. (1985). Host for phage and plasmid propagation was E. coli JM109. Compilation of sequence data, identity comparisons and gene bank searches were performed using a Beckman MicroGenie sequence analysis program. Calculations of evolutionary distances between flanking sequences of tRNA genes were done using the formula provided by Kimura (1977) with modifications according to van Ooyen et cd. (1979). (d) Transcription

in vitro

Nuclear extracts were prepared from HeLa S3 cells according to Dignam et al. (1983). Standard transcriptions were performed in lo-p1 volumes containing 12 mM-Hepes (pH 7.9), 12% (v/v) glycerol, 85 mM-KCl, 120 PM-EDTA, 300 PM-phenylmethylsulphonyl fluoride, 300pM-dithiothreitol, 10 mM-creatine phosphate, 5 mm-MgCl,, 600 pM each 14 /IM-GTP, 7.4 x lo6 Bg ATP, CTP and UTP, [w~‘P]GTP per ml, and 1.8 to 2.4 mg protein/ml. tDNA template concentration was adjusted to 3.2 pmol/ml, and a total DNA concentration of 80 pg/ml was achieved by addition of pUCl9 DNA. Both concentrations represent an optimal or nearly optimal value for all of the 9 tRNA genes. The mixture was incubated for 60 min at 3O”C, and the reaction was terminated by addition of 40 ~1 of 125 miw-sodium acetate (pH 5.5). 0.25% (w/v) sodium dodecyl sulphate, and 0.63 mg poly(U)/ml. RNA was extracted t,wice with phenol, once with chloroform/ isoamyl alcohol (24 : 1, v/v), precipitated with ethanol and analysed on 10% (w/v) polyacrylamide/8 M-urea gels (20 cm long. @3 mm thick). Gels were autoradiographed for 16 to 20 h at - 80°C on preflashed X-ray film in the presence of an intensifying screen. Autoradiographs were evaluated densitometrically using an Elscript 400 scanner. Preparative transcriptions were performed in 50 or 100~~1 portions as described above, with the exception that the concentrations of GTP and [w~‘P]GTP were 3 PM and 3.7 x lo7 Bg per ml, respectively. Precipitation of synthesized RNA was performed in the absence of sodium dodecyl sulphate and poly(U) and the products were separated on a 10% polyacrylamide/8 M-urea gel (40 cm long, 1 mm thick). RNA bands localized by autoradiography were excised and eluted with vigorous shaking overnight at 4OO”C in 300 to 500 ~1 of 10 mM-Tris. HCl (pH 7.5). 300 mM-NaCl, 1 mM-EDTA. 1 y0 phenol. The eluted material was extracted twice with phenol and once with chloroform/isoamyl alcohol (24 : 1, v/v), precipitated with ethanol, washed and dissolved in 10 to 100 ~1 of water. (e) Analysis

of in vitro

transcription

products

Up to approx. 3.3 x lo4 &s/s of RNA transcript digested with RNase T, and RNase A, respectively,

were in the

507

presence of 5 pg of yeast tRNA, essentially as described by Silberklang et al. (1979). In order to identify the 5’-phosphorylilted termini of transcription products, portions of RNA transcripts were treated with calf intestinal alkaline phosphatase before digestion with RNase as described by van To1 et al. (1987). Fingerprint analyses of untreated or dephosphorylated transcripts or processing products were performed according to Silberklang et al. (1979). Oligonucleotide sequences were derived from the known tDNA sequences and from the location of the oligonucleotides in the fingerprints (Domdey et al., 1978). (f) Primer

extension

analysis

of tDNA

transcripts

5’-Labelling of oligonucleotides was performed with [Y-~~P]ATP as described by Chaconas & van de Sande (1980). Pre-tRNAs were synthesized by preparative tranabove in 25-~1 portions. scription as described 32P-labelled pentadecadeoxynuc2.5 x lo5 &s/i of leotide (approx. 7.5 ng) were added to the phenol/chloroform-extracted transcription products and the mixture was precipitated with ethanol. For hybridization of the primer to the transcripts, the dried pellet was resuspended in 10 ~1 of 10 mM-Tris.HCl (pH %O), 250 m&f-NaCl, 1 mmEDTA, heated to 90°C for 5 min, chilled on ice for 10 min, and incubated at room temperature for about 10 min. The hybridization mixture was supplemented with 15 ~1 of AMV reverse transcription buffer (25 mm Tris. HCl (pH 8.0), 16 mM-MgCl,, 8 mirr-dithiothreitol, @4 mM each dATP, dCTP and dTTP, @8 mM-dGTP, @l unit reverse transcriptase/ml) and reverse transcription was performed at 37°C for 1 h. Then 5 ~1 of poly(A) (@5 mg/ml) in 1 M-sodium acetate (pH 54) was added and the RNA-DNA hybrids were collected by precipitation with ethanol. The products were electrophoresed on a 20% polyacrylamide/8 M-urea gel (40 cm long, 1 mm thick). Bands were excised after autoradiography and nucleic acids were eluted in 200 to 300 ~1 of 20 mM-Tris.HCl (pH 7.5), 0.1 mM-EDTA by vigorous shaking at room temperature, followed by extraction with phenol and chloroform/isoamyl alcohol (24 : 1. v/v) and precipitation with ethanol. The elongation products were submitted to chemical DNA sequence analysis essentially as described by Maxam & Gilbert (1977). The cleavage products were analysed on a 20% polyacrylamide/8 Murea gel (40 cm long, @3 mm thick).

3. Results (a) Sequence of human tRNA”“’

genes

The sequences of five newly characterized tRNA genes are shown in Figure 1. Figure 7 shows the precursors derived from all nine known tRNAVa’ genes in cloverleaf structures. The nucleotide sequences written in enlarged letters represent the respective tRNAs. pHtV1 (Arnold et al., 1986), pHtV6 and pHtV8 (Fig. 7(a), (f) and (h), respectively) code for the major tRNAT;A $ ecies, whereas hp pHtV3 (Arnold et al., 1986) encodes the minor tRNAz$ Both species differ only in the first position of the anticodon (position 34). The structural genes in the other five clones all show sequence variations as compared to the genes mentioned above, and hence do not code for known, functional occurring in the tRNAV”’ species. The mutations mature tRNA domains of pHtV2, pHtV5 and pHtV7 (Fig. 7(b), (e) and (g), respectively) do not

508

H.-U.

Thmnn

et al.

pHtV5 -365 GMTTCCCCC

-355 TTl'CCAGGGT

-265 GAGAMGCCT

AACAGAGGGA

GTACTTTMA

-165 CGCAGTATCT

-155 TCTl'TGTATG

ACTCGTGMC

-65

-55 CCAMTCTGG

GATATCCAGA

AAMTlTl'CT

ACGCGAMGG

56 TCCCCGGTTC

146 CGCACCATCT

TFAGCCGTTT

661 I 176 86 96 GMACCGGGC AGAAGCMCCTGTA-C GAGGMTCCT I 166 176 186 196 AGGAGAGMG GTPCTCCTTCTACCCTGTGG TGTGCATATA

246 TCCTGTTTTC

GA

-614 TCTAGAGCTC

-604 TACCAAACAA

-594 TGGMCAATA

-584 AAMTGGTCT

-574

-564

TGTGGGCTAC==CCTCmC

-514 ACTCACGAAC

-504 CCATGGCCTC

-494 ATATTCCCCA

-484 AGCAGCTTCT

-474 TCTGAAAGTCTC~CTC~A

-414 CCCACTGTCA

-404 ACCCCAGACA

-394 GCTCGACTGC

-384 CTCACGTCTC

-374 TCTGAGGTCG

-114 GGCATCAAAA

-304 -294 CTTCCTTTTC __ _ _ _ _ _ _ __ TTGCTTCTCT __ __

-214 TGCAGTGGCG

-204 CAGTCATAGC -104 ATCCCCATTA

‘ITCGATGTTA

-255

36 TTCGCCTCAC 136 AlTGCACCCC

236 CCAGAGCCCT

-345 TCMTCCGTG

46

-335 CCTCCGGCTA

-325 TTlTCTTGTG

-315 GTAAmTCT

-305 GCCTCCCAAG

-235

-225 TCTACTGGGG

-215 GGCTGTGTM

MTlTCTGTG

-125

-115

-245 WTGTCC

-145

-135 TCCGATATTA

-45

CTGATCCFAATAMTAGGM

-35

156

-205

-25

-265 CACAGAGCCT

-195

-165 MAGMMTT

CTGTGTTTGA

-105 AGCTTATCM

-15

-95

-5

Ill CAGGGCTKT

-275 TCTGCTfXTG -175 TTCCCCMGT

-85

CCTTGATMT

TATCAMTGT

TCTTCTCCTAGGAGAMMC

-295 AGATGGAGAT

TTCCTGAAAG

I

-75 MMGATCM

16

26

GTAGTGTAGT

GGlTATCACG

TAGCTGMTT

116 TCTCAGTGCA

CGTTTI’TAGA

206 MCCGAAGTA

CACCTGCCGC

106

126

216

226 CTTTCTCCCA

RHtV6

-114 CTC -14 GGTGCGCCAG

-4 GAMCGCGTG

87

-554 ATTCCACTCC

AGTTTCTCCA

GGCACACMG

-524 CTCCAGCTCC

-464

-454 GMMCGCTG

-444 CCAGCGCGCC

-434 CCTGCTGTTC

-424 MCCCCCAGG

-364 CCACCACACC

-354 TTCTGTCAGG

-344 ACTCCCATGC

-334 CCGCTTATGC

-324 CACCACATCG

-284 CTCTCTCTCT

-264 -274 CCCTTTTTCTTTCTTTTCTC

-254 TCTTTCGAGA

-244 CTGAGTCTTG

-234 CTCTGTCTTC

CAGGCTGGAG

-194 TTACTGCAGC

-184 TGCAGCCTCG

-174 ACCTCCTGGG

-164 CTCCAGCCGT

-154 CCATCCATCT

-144 CAGCCTTCCA

-134 AGTGCTGMA

-124 CGTCCTTTTC ___ __ __ _ _ _

-94 GCCAAGCTCG

-04 MGGGACGCA

-74 TGGAGGMGC

TCAGAATTAC

-34 CTTGTGTGGT

B -24 GGCTTATTGT

A

11

7

-64

CMGTTTCCG

17 27 TAGTGTAGTG GTTATCACGTTCGCCTAACA

-54 CATGAGMAT

37

-544

-44 AAMTGCGGC

47

-534

-224

CGCGMAGGT

57 67 CCCCGGTTCG MACCGGGCG

GAM’XGAA’E

I

77

177 GGCCATCMG

97

CDTATGCTTT -

TCATCTCATT

107 ACTTCAMTT

117 TATTACMGA

127 137 AMTTAGCCT GGAACACCCC

147 ACCACTATCT

157 CCACCTGGTT

167 ACGGAGAMA

187 GGTATTTATA

197 CCGTTGCCTT

207 TCCTCAACGG

217 TTGAGGCACC

227 237 TGGGAAGAAC TGMCCCACC

247 TCACTTGCCC

257 TGGACAGCGG

CGCCGACGM

CTTCGCACGA

287 ACCCCGTCCA

297 CTGGACGCTG

307 CGCGCTGCCA

317 AGTGGMTCC

327 337 AGATCCCGAG CATGCGTGGG

347 GCCCMGGCG

357 AGTTCCAATG

367 MTCCTCCTT

TTGCMCCCA

387 TGGGGGGACA

397 AAATGGAGM

407 ATCACMCM

417 GTTGGCAGAG

GTGGMTTC

267

277 377

pHtV7 - 4-4 8 -438 TCGAGCCTAG GCGACAGMC

-428 GCAGACTCTC

-418 TCTJ,AMM,T

-408 ACAAPAAACA

AMCACACM

-388 AAMGTAGW

-378 TGATCTAAAG

-368 CAMTGGTGA

-358 ATATCTGCAG

-348 AAACTMTGT

-338 GATATCAAGT

-328 ATAGATTGAT

-318 ATCTATGAGA

-308 TCMAAGCTT

-298 CATTTGACCC

-288 CGGATGGGTT

-278 TGTGGGCCGT

-268 TTGACGTGCA

-258 TTGTTTTAAT

-248

-238 TTTATGATAA

-228 AGTGATAMC

-218 ATGTGTGTGG

-208 GTGGGTGGGT

-198

GGAAGGTGGA

GTGTTGGAM

-188 GGGTGTTTAT

-178 TGGATTTCTT

-168 TTTGTCTTCC

TTATTTTCCT

-148 TCTTTCGGTC

-138 TCTACTGTAT

-128 CAAATACAAT

-118 AACAGATATT

-108 CAGGAAGAGC

-98 GGATATTGCT

-88 TTAGCTGACT

-78 GTAGCCAGTG

-68 TTTCTTTGGT

-58 GGGACAACCC

-48 MCTATCACT

-38 GCAACATTAT

-28 CTCTATAGGA

GAATTTAAAG

53 AGGTCCCCGG

ti 63 TTCGATCCCG

I 731 83 93 103 GGCGGAAACA GGTCAGCTGT TTTTCCTMC CGGAGAGTM

153 TCTTTCTCTT

163 CCGCCTTCCT

173 CCTGCCTTTT

183 AGACATTTTT

253 AAGGCCGMG

263 AGGCCGGATC

273 ACCTGMGTC

353

363 ATCTCTACTA

373

463 GGTGGTGGTT

ACCCCACCCC

453 GAACCTGGGA

-398

-158

TCCGTAGTGT

i 23 t 33 43 AGCGGTTATC ACATTCGCCT CACACGCGAA

113 TACTATTTGA

123 MTTCACCGA

133 AAGAACGATT

143 CTAGTCCCAT

223 TGGCTCACGC

233 CTGTMTCCC

243 AGCACTTTGA

283 AGAGTTCGAG

213 CCGTGATTGA il 293 303 313 ACCAGCCTGG CCUTGGT GAACCGCCCC

323 CCCCTCCCCC

ACCCCGCCCC

ACCCCGCCCC

AMATACMA

383 AATTAGATGG

393 403 GCGTGGTGGC GTGCGCTTGT

MTCACAGCT

423 ACTTGGGAGG

433 CTGAGGCAGG

443 AGMTCGTTT

473 GCAGTGAGCG

483 GAGATCACGC

493 503 CACTGCACTC CAGCCTGGGG

GACCGAGCGA

523 GACTCCGTCT

CMAACTAM

-18

-8 AATTATGACG

193 ATGGGATTTA

(1 3 CCTACCGGTT

13

203 MAAAATTTT _-_-----

553 CAAACTCGCA

Fig. 1.

413 513

333

343

533

543 ATGE@X-tT

A Human tRNA Gene Family

pl-itvt3 -517 ATGCTCGCTA

-507 G~~~mTK;~A'f

-417

-407

CACTGCAAGC

TCCGCATGCC

-317 TTTAGTAGAG

-497

-487 TTATTA'J-l'AT

-477 TATTCCAGAC

-467 GGAGTCTCGC

-457 TGTGTCGCCC

AGGCTGCAGT

-437 GCAGTGGCGC

-427 GATCTCGGCT

-397

-307

GGG'ITCACGC

CATTCTCCTG

-377 CCTCAGCCTC

-367 CCGAGTGGAC

-357 TACAGCGGCC

-347 GCCACCACGC

-337 CCGGATAATT

-327 TTITTGTATT

-257 ATCCGCCAGC

-247 CTCGGCCTCC

-237 CAAAGTGCTG

-227 GGA'ITACAGG

-137 TTl'GACAMC

-127 GGCARACGC

-37

-27 CAC'ITATAAA

A

-307

-297

-287

-277

-267

ACGGGGTTTC

ACCATTTTAG

CCAGGATGGT

CTCGATCTCC

TGACCTCGTC

-217

-207

CGTGAACCAC

CGCGCCCGGC

-197

-187

-177

-167

CTTGTl'TTGC

TTlTMTTGC

TGGAAGTGAC

TGGGGTACCC

-157 TTAGAGGTCA

-147 CGCTTTACAC

GTTCTGGGGA

-87 AGAATGGATC

-77 TAAGGGCCAT

-67 GACTCCATGG

-57 GTGMGCCGA

CACCTGGATG

14 CCGTAGTGTA

-117----------;7 AGGGCCACAA

-17

GGAGACTGGT

-447

-7

h

4

-47

TTGACTATM

GCATCTGTTT I 84 94 104 GACTGTGTTT TCCTTCCAGT TCAAAAAGGT

114 TTCTTAGTGT

24 34 44 54 64 7'4 GTGGTTATCA CGTTCGCCTA ACACGCGAAA GGTCCCCGGT TCGAAACCGG GCGGAAACAA 1 124 134 144 154 164 174 GGAGTCTATG TGTATAAACG TTCACMGTT TTGCTACACT TAGACACCGG CCGGAl-l-lTC!

184 CAATCCATTG

194 CCMTTAACT

204 TCMCCTGCG

214 TATTCCATGT

224 CTACGTCTGT

284 CTGCAAAGTC

294 CCGGCGGATC

304 AAATACAGTC

AGCCGCCGGG

GMTGCCACG

384 CTTGGTGGM

394 GTTCACGCCG

TGGMGGTGT

414 GCACCGGCGG

404 CCCTCCGCTT

494 CCTGCAGAGG

504 MCCTMCCA

584 GACGGAGCM

594 MGGACAG,X

MCGG-G

684 GCCTGCAGCA

694 CTGACACGGC

784 ACCCATCCTC

794 ACmGCAGCT

884 TGCTGCAAAG

GCTTCAGAGA

AGMGCCGGA

-249 GGCCGAAGM

-239 CGMGGACCG

-229 TTCACAGAl-l'

-219 AMTTAMGT

-199 -209 CTCTATPGTGTTCTPCCCTA

149 ACACTGGAGG

-139 GGCTGGGTCT

-129 GAGCTMTCA

-119 TlCG'lTlTTC

TGGTlTGAGAGGTCGGTACA

-49 AAGAAACACG

-39 GMCTAGGCA

GGMGMCTA

-19 TTTACTCTGA

AAMTAMCC

MGGTCCCCG

62 GTTCGAAACC

72 GGGCGGAMC

I ACG zcz&s

152 CATAAATTTC

162 CTCTCTTCTT

172 GTCTTGCTAG

182 ATCGGGGCAG

AGTCATAGAG

TAACCTCTCC

894

314

234 MTATGAGGG

324

244 TGAGAAGTM

334

264 TGGTCATTAT

274 TAGGGTCTK

354

TGAACGATTA

AAAAGTGCM

364 GAAGGCTCGG

374 GTTGGAAATC

424 434 GCCAATAGTG ATGCCGGTM

444 CTACMCGTG

454 TACCACACAT

464 CMGTAGTCT

GCAGGTGTGG

514 TTCTGGGACG

524 534 TTGTTTACAC ATGAGGCCAC

544 TGAGGCATGG

554 ACAGGTCACA

564 CMCGCTGCG

574 GMCACGTAG

614 -CAGGGCA

624 634 CAGAGAMT-" CMGTGMAT

644 ACAGGCTGGT

654 TCAGGAGGAG

664 GACTGCGCCA

674 CTTCGGGCGG

704 mGGGmC

714 GCAGTGGCGG

724 734 TGGAGGGCGGTCMGAGGAC

744 CATCAGCCTC

754 TCCMGGCGT

764 GACCGTCGGC

774 CTCCCTCTGC

804 CGTCGGCI-K

814 GGGCCGTGGG

a34 824 ACGCGGCGGGTGAGTTGGGA

044 GCCCGATCCC

a54 CAGGGGCCTC

864 TTCAGCMTC

074 TCAGGGAAAA

904

914 GGGGTGGGCG

GGATCC

404

604

GMTGACAGA

344

254 GTGGTCTTAC

414

pHtV9

52

-29

-109

-99

11 CTI'AGTGGGT I 92 102 CCKATCTTACTACGMA

TTCAGTCATT

-179 TGTAGMTM

-169 ATKTATGGC

-159 TTCAGTTTGA

-89 GAGACGCACG

-79 CGCACATCAG

-69 CAMACACGC

-59 ACTTGGAACG

TAGTGGTTAT

I32 CACCl-l'CACC

42 TCACACGCGA

122 GTGTATCTTT

132

TTCTTCCAGT

TAMGACTlT

142 lTCTGGCTGT

212 ACAGCCTCAC

222 ACACGTATCG

232 CCAGGGGCAG

MCCAGCT

-189

12

-9

202 192 CAGTCCTTAG GCCGGGCCTC

TTCCGTAGTG 112

22

Figure 1. Nucleotide sequences of 5 human tRNAV”’ genes and flanking regions (non-coding, tRNA-like strands). The structural genes are boxed and the putative oligo(T) termination signals are doubly underlined. Sequence variations as compared to the known mammalian tRNAVs’ isoacceptors (not regarding the sequence difference at position 34) are marked by arrows. Ah repetitive elements are underlined and bordered by shadowed arrowheads; flanking direct repeats are indicated by broken lines. pHtV5, This clone was isolated from a human genomic library in lambda Charon 4A (see Materials and Methods). pHtV6, A 65 kb EcoRI fragment containing this tRNA gene was isolated by screening a genomic library in lambda gtWES/lambda B representing fragment population 1 (see Materials and Methods). The tRNA gene-containing 1049 bp EcoRI-XbaI fragment was subcloned in pUC19 and sequenced. The region of homology with pHtV3 is indicated by filled arrowheads. pHtV7, An 8 kb EcoRI fragment with a tRNA gene was found during screening of a lambda gtWES/lambda B library representing fragment population 2 (see Materials and Methods). The tRNA genecontaining 1.7 kb PatI fragment was subcloned in pUC19 and partially sequenced using the E~oIII/E~oVII method (Yanish-Perron et al., 1985). pHtV8, This tRNA gene on a 9 kb EcoRI fragment was detected together with pHtV7 by screening the same gene library. A 2.0 kb BamHI fragment subcloned in pUC19 was partially sequenced using the ExoTIT/EzoVII method. pHtV9, A 15 kb EcoRI fragment carrying a tRNAVa’ gene was detected during screening a lambda EMBL4 library representing fragment population 3 (see Materials and Methods). A 1.1 kb Pat1 fragment subcloned in pUC19 was partially sequenced. The sequences of pHtV1 to pHtV4 have been published (Arnold et al., 1986; Kahnt et al., 1989). It should be noted that, due to typing errors, 3 mistakes have been published: nucleotide 83 is C instead of A in pHtV2 (Fig. 3(b) in Arnold et al., 1986), and a C has to be inserted between nucleotides - 115/- 114 and between nucleotides - 136/- 135, respectively, in pHtV3 (Fig. 3(c) in Arnold et al., 1986).

510

H.-U.

Thowmnn et al. I----

pHtV3

~ECOAI

TECoR'

pmm

(a) TCATACCCATTA CCMGCTCAMGGGACCCACGGMGMGCTCAGMCTC~ATGAG~T~TACCGCCGTGTGTCG~ACTM~ATGA III1 lllllll IIIIIIII IIIIIII II Ill ll::IIIIIII I IIIIlIIIIIIIII I III Illll TCATGCCCATTAGCCMGCTCGM~ACGCA~GAGGMGCTCAGM~ACCATGAGAMT~TGC~CC~GTG~G~GC~A~GT~

III

II

III

II

TGGCAGAAAAGCATCMQCC III

I

I

I

T~TA~~ACTTMCTGC~C~~A~ACACCG~TA~ATACCG~C~~CCGCCGCMTCA~GCATCTGGGMGMCGGMCC~A~~ I I :IIIlll l I I IIIIIIIlIIIIIIII TATOC~TCATCNATTACTTCMA~ATTACA

IIIIIIIlIIIIIIIIIIIIIIIII

~IIIII~:l::

I

AGAAGTATTTATACCGTTGCCTTTCCTCM~G~

IIIIIIIII

TGA~&~GGGAAGMCTGAACCCACC

TCACTTGCTGTTGACACCAGC ATCACGMCNCCCAC~~CCGTCCAG~GATGGA~ACTAGCCTAGMCA~CCACCMTATCTCCACCCACTTA lII:lllI I IIII I II llllllll I III IIIIIIIIIII IIII IIll: IIIIIII III:lIIIII TC~W~‘WCCTQGACAGCQGCGCCGACGMCTTCQCACQMQCCCG~CACTQQA c&&~,&&GGAAcAccccAccAcTATCTCCACCTGGTTA AGGAGAAAAGGCTATCMGGGCACMTGCCAAATGGMTTC IIlIIIIIIII 1111111 II I llllll llllll CGGAGAMAGGCCATCMGGGCGCGC~CMG~M~C

III

l

(b)

Figure 2. Comparison of pHtV3 and pHtV6. (a) A representation of the EcoRI fragment and the XbaI-EcoRI fragment of human DNA contained in pHtV3 and pHtV6, respectively. The 4-fold direct repeat of pHtV3 (see Arnold et aE.,1986) is symbolized by 4 small open boxes and the truncated A&u element of pHtV6 (Fig. 1) by 1 big open box; broad filled arrowheads mark the direct repeats flanking the Ah element. tRNA coding regions are represented by filled boxes. Hatched boxes indicate the region of sequence homology between the 2 loci. (b) Alignment of parts of the DNA fragments contained in pHtV3 and pHtV6. Exactly those segments are shown that are represented by hatched boxes in (a). Structural genes (non-coding strands) are boxed and nucleotide conservation is indicated by vertical lines.

appear to interfere with the secondary or tertiary structure of the resulting tRNAs, nor do they affect conserved sequences in any case. For instance, in the altered acceptor stem of the pHtV5derived tRNA, base-pairing is fully maintained (C. G pair, positions 2 and 71, respectively, and U. A pair, positions 6 and 67, respectively; Fig. 7(e)). Four mutations occur in the structural gene of pHtV2 (Arnold et al., 1986)) resulting in a U. A pair (positions 51 and 63, respectively) and an A nucleotide each in position 54 and 57. The U to A transversion (position 54) destroys the tRNAVa’-specific sixth base-pair in the T$C stem (Jank et al., 1977a), but an intact T$C stem with five base-pairs, as in all other tRNAs, can still be formed. It is more striking that A54 is normally found only in initiator tRNA”“’ species and, moreover, that a U51. A63 base-pair that occurs in all initiator tRNAs is present also in this tRNA (Sprinzl et al., 1989). In the tRNA of pHtV7, a U to C transition (position 60) also destroys only the sixth tRNAVa’-specific base-pair (Figs 1 and 7(g)). In contrast, some of the sequence variations occurring in the structural genes of pHtV4 and pHtV9 are expected to impair tRNA secondary structure. In pHtV4 (Kahnt et al., 1989), the G65 replacement by A abolishes complementarity with C49, thus altering the structure of the T$& stem (Fig. 7(d)). While the arm might still be stable because of the extra base-pair between

T54 and A60, the tertiary structure of the tRNAlike product should be seriously affected. The other mutations in this gene lead to G. U instead of G. C pairs, which often occur in tRNAs (Sprinzl et al., 1989). In pHtV9, the G to A transition at position 30 destroys the fourth base-pair of the anticodon stem and, presumably, the structure of the arm altogether (Figs 1 and 7(i)). (b) Flanking

regions

The tRNAy;h g ene of pHtV6 (Fig. 1) shows remarkable similarity in its flanking sequences with those of the tRNAE& gene of pHtV3 (Arnold et al., 1986). There is 77 y. and 78% identity for the 5’ and 3’ flanking region, respectively. An alignment of the two sequences is shown in Figure 2. The identity spans the whole known sequence downstream from the structural gene of pHtV3 as far as the natural EcoRI site. Hence, it remains undetermined whether there is still further similarity between these two loci. Upstream from the tRNA gene, the identity ceases abruptly at nucleotide - 120. This coincides with the border of an Alu repeat in the 5’ flank of the tRNA,V,“:: g ene of pHtV6. In spite of short oligonucleotide stretches shared between pairs of the remaining clones, no significant similarities were found, and the maximal overall identity

obtained

by

alignment

of

flanking

A Human tRNA

(a)

Gene Family

(b)

(e)

Figure 3. Transcription of tRNA”” genes. All tRNA”“’ genes disscussed are compared with respect to their transcription pattern at’ 1 mM and 5 mM-MgCl,, respectively (see Materials and Methods). (a) to (c): transcription products of pHtV1 to pHtV3; (d) to (h): transcription products of pHtV5 to pHtV9. The transcription of pHtV4 is reported elsewhere (Kahnt et al., 1989). The products derived from pHtV3, pHtV5 and pHtV9 are overexposed to visualize all bands. Lanes 1 each show the RNAs obtained after transcription for 60 min at 1 miw-Mgcl, in a volume of 5 ~1; lanes 2 to 5 each show the products of a pulse chase transcription experiment performed at 5 mM-Mgcl, and in a by extraction with phenol volume of 30 ~1. After 15 min and 60 min, 5-~1 portions were removed and deproteinized (lanes S and 3). To the remaining 20-~1 reaction volume, @5 ~1 of cr-amanitin (10 mg/ml) was added to a final concentration of 240 pg/ml, and 5-~1 portions were removed after an additional 20 min and 40 min, respectively (total incubation time of 80 and 100 min, respectively; lanes 4 and 5). The products were analysed on a 10:~ polyacrylamide/8 M-urea gel. Roman numbers identify primary transcripts, processing intermediates and the mature tR,NA (see Results).

sequences rarely exceeded 50%. Even within the immediate neighbourhood of the structural genes, i.e. 50 bp upstream and downstream, no significant homology was detected that might be expected at least as the result of functional restraints. There is no obvious regularity concerning the level of sequence identity between groups of genes; e.g. welltranscribed genes do not share an overproportionally high degree of sequence conservation as compared with less efficiently transcribed genes or vice versa.

Surprisingly, there is a striking homology between the 5’ flanks of the structural gene encoded in pHtV5 (Fig. 1) and those of the D. melanogaster tRNAz”’ gene (Addison et al., 1982) and between the 5’ flanks of the gene encoded by pHtV7 and those of a D. melanogaster tRNA:E’ gene (DeLotto & Schedl, 1984). The sequences of the structural genes themselves share an identity of only about SS%, but the 5’ flanks show about 60% conservation within a nucleotides -27 and -44, region spanning respectively.

H.-U.

Thomann et al.

f

Figure 4. RNase T, and RNese A fingerprint analyses of tRNAVa’ genes. Selected fingerprints of the tRNAV”’ gene products characterize the primary transcripts, their processing intermediates and mature tRNA, and hence explain the sequence of processing events. Al, RNase T, finger rint of dephosphorylated. pHtVl-derived, [w3*PJGTP-labelled RNA I from Fig. 3(a); Bl, RNase T, fingerprint of [a- 8 P]UTP-labelled RNA I from Fig. 3(a); Cl, RNase T fingerprint of [a-32P]GTP-labelled RNA II from Fig. 3(a); Dl, RNase T, fingerprint of [a-32P]GTP-labelled R&A III from Fig. 3(a): El, RNase T, fingerprint of [a-32P]GTP-labelled RNA IV from Fig. 3(a); A2, RNase T, fingerprint of pHtV2-derived, [cr-32P]UTP-labelled RNA II from Fig. 3(b); B2, RNase T, fingerprint of the dephosphorylated,

A Human

Like many other tRNA genes (Buckland et al., 1983; O’Neill et al., 1985; Pirtle et al., 1986; Doran et al., 1987), six of the tRNAVal genes have complete or truncated Ah elements in their neighbourhood.

(c) Transcription

513

tRNA Gene Family

in vitro

To study the transcriptional activity of the genes, plasmid DNA was incubated in a HeLa cell nuclear extract (Dignam et al., 1983) as described in Materials and Methods. As shown in Figure 3, each of the genes is active in vitro. Their transcriptional activity is strongly dependent on the concentration of magnesium ions in the reaction mixture (not shown). The majority of the genes reaches the maximum of transcriptional activity at 2 mM or 3 miw-Mg 2+ . There is one exception; pHtV1 shows the highest transcriptional efficiency at 4 mM-Mg2+, but then stays remarkably active until 8 mM, when all the other genes hardly produce detectable transcripts. If the maximal transcription efficiency of pHtV1, which is the most efficient of all genes, is taken as a 100% standard, pHtV6 and pHtV8, the other clones encoding tRNAz& reach 59% and 79% at 3 miw-Mg2+, respectively. pHtV3, the only gene for tRNA,-,, Va’ found so far, has a peak efficiency of 18% at 3 mM-Mg’+. Surprisingly, this gene, although it apparently codes for a functional tRNA, is one of the least active, together with the severely altered pHtV9 (17% at 2 m&f-Mgcl,). In contrast, the other considerably altered genes pHtV2 and pHtV4 are nevertheless transcribed with 83% and 47 o/;, efficiency at 2 mM-Mgcl,, respectively. Finally, pHtV5 shows average transcriptional activity (40% at 2 mM-Mg2+) and pHtV7 is nearly as active as pHtV1 (87% at 3 mM-Mg2+). In contrast to the transcription efficiencies, the enzymes responsible for processing appear to have maximal activity between 4 mM and 8 mM-Mg2+. Tn order to discriminate between tRNA precursors, processing intermediates and mature tRNA. the transcription was carried therefore out at 1 mM-MgCl, and 5 mm-Mgcl,, respectively (Fig. 3). Essentially the same patterns of RNA bands occur during transcription at 1 mM-MgCl, for 60 minutes (Fig. 3, lanes 1) and within the first 15 minutes of

transcription at 5 mM-MgCl, (Fig. 3, lanes 2). When transcription at 5 mM-Mg2+ continues for an additional 45 minutes, maturation products are formed, with the exception of pHtV4 (Kahnt et al., 1989) and pHtV9 (Figs 1 and 3(h), lane 3). The addition of a-amanitin to a final concentration of 240 pg/ml after 60 minut’es transcript’ion at, 5 m-n-Mg2+ confirms that, pre-tRNAs are processed to mature tRNAs 2%~ intermediates (Fig. 3 (a) to (g), lanes 4 and 5) with the exception of pHtV4 (Kahnt et al.. 1989) and pHtV9 (Fig. 3(h), lanes 4 and 5). The shortest processed RNA band of pHtVX-derived transcripts (Fig. 3(b), lanes 3 to 5) is a few nucaleotides longer than the mature tRKAsVa’ of the other clones. This pre-tRNA is processed correctly at its 5’ end, whereas the 3’ end is not completely processed (see below). As expected, the 3’-terminal (:(‘A sequence is not added to this 3’ end. but is to th(h mature 3’ ends of t,RNAs derived from all the other clones. When isolat,ed pre-tRNAs were incubated again in HeLa cell nuclear extract. but without ATP and CTP, the resuking tRNAs had the correct length of 73 nucleotides for pHtV1, pHtV3 and pHtV5 to pHtV8. Cpon addition of ATT’ and CTP. these tRNAs were converted into mature t,RNAs 1)) addition of t’he 3’-terminal (ICA. whereas the corresponding band of pHtV2 remained unc*hangetl (not shown). (d) Fingerprint

analyses of transcription processing products

and

Fingerprint analyses were performed as described by Silberklang et al. (1979). In all cases RNase T,. and in some cases RNase A, digestions were used to determine the structure of all major bands produced at 5 mM-Mgcl,. (i) pHtV7

(Arnold

et al., 1986)

The transcription pattern of pHtV1 shows four RNA bands of different size (Fig. 3(a)). The fingerprint of RNA I indicates an RNA molecule with flanking sequences at its 5’ and 3’ ends. Only RNA I is produced and no significant processing takes place at 1 mM-Mgcl,. Figure 4 Al represents the RNase T, fingerprint of phosphatase-treated RNA I. In addition to the pattern expected for the mature

[a-32P]GTP-labelled RNA II from Fig. 3(b): C2, RNase A fingerprint of the dephosphorylated, [cc-32P]GTP-labelled R’NA II from Fig. 3(b); D2, RNase A fingerprint of the dephosphorylated, [a-32P]GTP-labelled RlrjA III from Fig. 3(b); A3. RNase T, fingerprint of pHtV3-derived, [a-32P]GTP-labelled RNA ITT from Fig. 3(c); B3, RNase T, fingerprint of [cr-32P]GTP-labelled RNA IV from Fig. 3(c); A5, RNase T, fingerprint of pHtV5-derived, [a-32P]UTP-labelled RNA I from Fig. 3(d); B5, RNase T, fingerprint of the dephosphorylated, [a-32P]GTP-labelled RKA I from Fig. 3(d); A6. RXase T, fingerprint of pHtV6-derived, [a-32P]UTP-labelled RNA I from Fig. 3(e); A7, RNase T, fingerprint of pHtVT-derived, [cc-32P]GTP-labelled RNA I from Fig. 3(f); B7, RNase T, fingerprint of [cc-32P]GTP-labelled RNA IV from Fig. 3(f); (17, RNase A fingerprint of [a-32P]GTP-labelled RNA 1: from Fig. 3(f); AS, RNase T, fingerprint ot pHtV%derived. [a-32P]GTP-labelled RNA I from Fig. 3(g); BE!, RNase T, fingerprint of the dephosphorylated, [a-32P]GTP-labelled RNA III f rom Fig. 3(g); A9, RNase T, fingerprint of pHtV9-derived, [cc-32P]GTP-labelled RNA T from Fig. 3(h). The spots are identified by their sequence; 5’ phosphate and 3’ OH groups are indicated, 3’ phosphate groups are omitted. () Fragments deriving from the 5’ leader sequence only: (- -) fragments deriving from the 5’ leader and from the tRNA; (----) fragments deriving from the 3’ trailer sequence only; (---) deriving from the 3’ trailer and from the tRNA; (*) oligonucleotide containing m’G in a ring-opened form (Hagenbiichle rt al.. 1979); the fragments with intact m’G were characterized by further analysis (RNase T, digestion and cellulose thin-layer chromatography; Stange & Beier, 1987); X. position of the xylene cyan01 dye.

514

H.-U. Thowmnn et al.

domain of the tRNA (Fig. 7(a)), some oligonucleotides that originate from the transcribed 5’ and 3’-flanking sequences are present. ACCG, UUG and G derive from the 5’ flank; the latter derives also from the mature tRNA. If RNA I (Fig. 3(a)) is cleaved by RNase T, without phosphatase treatment, two new spots occur in the fingerprint, pG and pACCG, which derive from partial dephosphorylation of 5’ triphosphate ends in the extract and are not visible in Figure 4 Al. These findings suggest two initiation sites for transcription, G at position -4 and A at position - 7. Hence, the 5’ flank initiated at G ( -4) produces pG and UUG, whereas initiation at A (-7) produces pACCG and UUG. The 3’ flank of the pre-tRNA is represented by two AG fragments, one UCG and one AAACAAAG. The first five residues of AAACAAAG derive from the mature tRNA domain, the last three from the 3’ trailer. The site of exact transcription termination of RNA I (Fig. 3(a)) was determined by fingerprint analysis of the [a-32P]UTP-labelled transcript. Fragments CG, UCCCCG and AAAG from the mature domain of the tRNA, UCG and AAACAAAG from the 3’ trailer are not labelled in this case. Figure 4 Bl shows an RNase T, fingerprint of the non-dephosphorylated, [cr-32P]UTP-labelled RNA I. The three newly occurring oligonucleotides CUU-OH, CUUU-OH and CUUUU-OH confirm that transcription terminates within the first two to four residues of an octathymidine sequence 11 bp downstream from the tRNA”“’ gene (Arnold et al., 1986; Fig. 7(a)). The two RNA bands II and III (Fig. 3(a), lanes 3 to 5, Fig, 4 Cl and Dl) are both 3’-processed products of RNA I. In both cases, the trailer-specific oligonucleotides AG, UCG and AAACAAAG are absent, whereas pG and UUG in RNA III and pACCG and UIJG in RNA II (Fig. 3(a)) still represent the leaders. Some nucleotide modifications are observed in the 3’-processed pre-tRNAs. A partial exchange of inosine (I) for adenosine in the first position of the AAC anticodon creates a new oligonucleotide ACACG, since RNase T, cleaves next to inosine. The new oligonucleotide CCUI does not appear in the fingerprint because it is not labelled with [u-~~P]GTP. Modifications are found at positions 46 (m7G) and 58 (m’A). RNase T, does not cleave at and therefore UCCCCG changes to m7G m7GUCCCCG. m’A and m7G both carry an additional positive charge and hence displace the corresoligonucleotides. Finally, RNA IV ponding (Fig. 3(a)) represents the 5’ and 3’-processed transcript of pHtV1. The leader-specific oligonucleotides pACCG and UUG have disappeared and pG, the first residue of the mature tRNA is now present in stoichiometric amounts. The RNase T, fingerprint of [c+32P]GTP-labelled RNA IV is shown in Figure 4 E 1. The primary transcripts, their initiation and termination sites as well as the sequence of processing events was similarly established for pHtV2, pHtV3 and pHtV5 to pHtV9 by fingerprint analyses (Fig. 4). Fingerprints of pHtV4-derived

products were presented by Kahnt et al. (1989). We find in all cases that 3’ processing precedes 5’ processing, since only processing intermediates with intact 5’ flanks and mature 3’ ends are formed. Besides this common property, there are a few peculiarities of certain genes that deserve special attention. (ii) pHtV2 (Arnold et al., 1986) Pre-tRNA products are represented by bands I and II (Fig. 3(b)) due to transcription termination at different sites, i.e. within CCTTCT and within TCTT 11 and 19 bp downstream from the gene, respectively (Arnold et al., 1986; Fig. 7(b)). RNA bands longer than RNA I are readthrough products created by termination 42 (at TCTCTATTT), 63 (at CTTTT) and 106 bp (at TTCTTTT) downstream, respectively. The fingerprints of RNA II are shown in Figure 4 A2 to C2. A transcription initiation site appears to be an A at - 6, due to the oligonucleotide AGGGU occurring in RNase A fingerprints (Fig. 4 C2 and D2). RNA band III (Fig. 3(b)) is a partially 3’-processed precursor (Fig. 4 D2). Fingerprints of RNA IV are identical with those of RNA III. RNA V (Fig. 3(b)) has a mature 5’ end, but the 3’ end is still incompletely maturated, since the RNase A fingerprint displays the same trailerspecific oligonucleotide AAGU as the pattern of RNA III (Fig. 4 D2). Base modifications occur as determined for RNAs II to IV derived from pHtV1 (Fig. 3(a); Fig. 4 Cl to El). (iii) pHtV3 (Arnold et al., 1986) The majority of the RNA synthesized at 1 miw-MgCl,, i.e. RNA I, is terminated within a tetrathymidine stretch 11 bp downstream from the gene (Arnold et al., 1986; Figs 3(c) and 7(c)). The correct transcription initiation site could not be determined by fingerprint analyses. RNA IT is a partially and RNA III (Fig. 4 A3) is a completely 3’-processed derivative of RNA I. The completely maturated tRNA is represented by RNA IV (Fig. 4 B3). Modifications occur as shown for pHtV1 and pHtV2-derived products, but, as expected, not in position 34. (iv) pHtV5 (Fig. 1) RNA I (Fig. 3(d)) is produced by transcription initiation at G (-6) and A (-3), and by termination within the first three thymidine bases of a pentathymidine stretch 7 bp downstream from the gene (Figs 1, 4 A5 and B5, and 7(e)). The 3’-maturated processing intermediate is represented by RNA II, and RNA III was identified as the mature tRNA. (v) pHtV6 (Fig. 1) An RNase T, fingerprint of RNA T (Fig. 3(e)) clearly revealed transcription termination within ATCTT 3 bp downstream from the structural gene (Figs 4 A6 and 7(f)). Initiation was determined to occur at A ( - 2). RNA II is the processing intermediate lacking the complete 3’ trailer and

A Human tRNA RNA III is the complete maturated transcript identical with RNA IV derived from pHtV1 (Figs 3(a) and 4 El). (vi) pHtV7 (Fig. 1) The major primary transcript RNA I is produced by transcription initiation at A ( -4) and presumably at C ( - 6), since the oligonucleotides pACCG and pCUACCG, respectively, were found in equal amounts (Figs 4 A7 and 7(g)). Termination occurs within the five consecutive thymidine bases 9 bp downstream from the gene (Fig. 7(g)). RNAs longer than RNA I are readthrough products terminating within ATTT and TTTT 35 and 97 bp downstream, respectively. RNA II is a partially 3’-maturated intermediate, cleaved somewhere between the sixth and the eighth nucleotide of the trailer sequence. The completely 3’-processed intermediates are represented by RNAs III and IV (Fig. 4 B7) and the mature tRNA by RNA V_(Fig. 4 (17). (vii) pHt V8 (Fig. 1) In the case of RNA I (Fig. 3(g)), transcription initiation was determined to occur at A (-4) and termination within four consecutive thymidine bases, 8 bp downstream from the structural gene (Figs 4 A8 and 7(h)). RNAs II and III (Fig. 4 B8) are partially 3’-processed and completely 3’-processed intermediates, respectively. The mature tRNA, i.e. RNA IV is identical with RNA IV derived from pHtV1 (Fig. 4 El) and with RNA III derived from pHtV6 (Fig. 3(e)).

Gene Family

515

(viii) pHtV9

(Fig. 1) Initiation occurs at A ( -5) and termination closely downstream from the gene within a nonathymidine stretch (RNA I in Figs 3(h), 4 A9 and 7(i)).

(e) Reverse transcription

of tRNA precursors

A 15mer deoxyoli+onucleotide complementary to the mature tRNA ” from positions 6 to 20A, AACCACTACACTACG, hybridizes without mismatch to all primary transcripts derived from the genes of pHtV1 to pHtV6, pHtV8 and pHtV9. pHtV7-derived pre-tRNA has a U to C mutation at position 16 of the mature tRNA domain and hence one A. C mismatch appears upon hybridization with the deoxyoligonucleotide. By reverse transcription of pre-tRNA/primer hybrids, the first newly synthesized nucleotides are complementary to the first five residues of the aminoacyl stem, and the additional nucleotides are complementary to the 5’ leader sequences of the precursors. The last nucleotide of the cDNA represents the complementary initiation nucleotide of the pre-tRNA. The nine tRNA genes were transcribed in 25-~1 volumes at 95 m&r-MgCl, to prevent any processing of precursors. Their pretRNAs were then reverse transcribed as described in Materials and Methods (Dingermann et al., 1987). Figure 5 shows the resulting cDNAs separated on a 20 y0 polyacrylamide/SM-urea gel. Lane 11 displays the products obtained by reverse transcription of

11

12

Figure 5. Reverse transcripts of tRNAVal precursors. pHtV1, pHtV2, pHtV3 (Arnold et al., 1986), pHtV4 (Kahnt et al.. 1989). pHtV5, pHtV6, pHtV7, pHtV8 and pHtV9 were transcribed at 95 m&r-MgGl, in a volume of 25 ~1 for 60 min (see Materials and Methods) and the isolated RNA products were reverse transcribed. Lanes 1 to 12 represent M-UEL gel. Lanes 1 to 9, autoradiographs of the resulting reverse transcripts separated on a 20% polyacrylamide/8 reverse transcripts of pre-tRNAs derived from pHtV1 to pHtV9, respectively; lane 10, reverse transcripts of unfractionated RNA contained in 15 ~1 of HeLa cell nuclear extract; lane 11, reverse transcripts of pure tRNAVa’ (Jank et al., 19776); lane 12, reverse transcripts of purified pHtV2-derived RNA V (see Fig. 3(b)). The position of the 5’ 32P-labelled pentadecadeoxynucleotide used as a primer for reverse transcription is indicated by the open arrow, the reverse transcript derived from mature tRNA”“’ is identified by the filled arrow. All fragments marked with asterisks were further analysed by sequence analysis according to Maxam & Gilbert (1977) (see also Fig. 6).

516

H.-U.

Thomann

et al.

respectively. An apparent initiation site at C (-2), the sequence ladder of the corresponding cDNA is shown in Figure 6(c), may be due either to initiation at -2 or to fast degradation of pre-tRNA, since pCG expected after RNase T, digestion of precursors was not detected in corresponding fingerprints. Upon transcription of the tRNA gene encoded by pHtV4 initiation occurs at three sites, i.e. at G each in position -3, -5 and -7 (Figs 5, lane 4 and 7(d)). Sequence analysis of the cDNA obtained after reverse transcription of purified RNA V (Fig. 5, lane 12) which is the shortest product obtained upon pHtV2 transcription, revealed a mature tRNA”“* 5’ end. This provides further evidence for a correct 5’ processing of pHtVX-derived precursors. (a)

(b)

(c)

(d)

(e)

Figure 6. Nucleotide

sequences of selected reverse transcription products. Sequences were determined according to Maxam & Gilbert (1977). Reverse transcripts whose sequence analyses are shown are marked by encircled asterisks in Fig. 5. (a) Sequence of the reverse transcript of mature tRNA”“* (Fig. 5, lane 11); (b) sequence of the reverse transcript of pHtV2-derived RNA V (Fig. 5, lane 2); (c) sequence of the reverse transcript of pHtV3derived RNA (Fig. 5, lane 3); (d) sequence of the reverse transcript of pHtV5derived RNA (Fig. 5, lane 5);

(e) sequence of the reverse transcript of pHtV9-derived RNA (Fig. 5, lane 9). The G, G/A, T/C and C cleavage reactions are shown from left to right. Corresponding

sequences that analysed

are complementary

pre-tRNAs

are indicated

to those of the

at the left of each line.

Sequences from the bottom to the arrow derive from the 5’ end of the mature tRNAsVa’.

purified tRNAz& The sequence ladder of the marked cDNA band ends with a C (Fig. 6(a)), which is the complementary nucleotide of the first G residue in tRNA”“‘. The same pattern was obtained upon reverse transcription of unfractionated RNA contained in 15 ~1 of HeLa cell nuclear extract (Fig. 5, lane 10). This provides evidence for the presence of endogenous tRNAV”‘. Reverse transcription of pHtV1 (Arnold et al., 1986) and pHtV7derived (Fig. 1) pre-tRNAs confirmed the initiation sites determined already by fingerprint analyses (Fig. 5, lanes 1 and 7, respectively; Figs 7(a) and (g), respectively). The tRNA genes encoded by pHtV2 (Arnold et al., 1986), pHtV5, pHtV6, pHtV8 and pHtV9) (Fig. 1) appear to initiate transcription at additional sites that were not detected by fingerprint analyses (Fig. 5, lanes 2, 5, 6, 8 and 9, respectively; Fig. 7(b), (e), (f), (h) and (i), respectively). Fingerprint analyses of transcripts derived from pHtV3 (Arnold et al., 1986) and pHtV4 (Kahnt et al., 1989), respectively, did not reveal defined sites of transcription initiation. However, the patterns of cDNAs obtained after reverse transcription of the respective precursors allowed a clear deduction of these sites (Fig. 5, lanes 3 and 4, respectively). In the case of pHtV3, initiation occurs mainly at A (-6) and in minor amounts at A (-5) and G (-4),

4. Discussion (a) Genomic

organization

of human

tRNA”“’

genes

tRNA genes in eukaryotes are often organized in multigene families (for a review, see Sharp et al., 1985). Around ten to 20 gene copies for each of the about 60 tRNA isoacceptor species in human cells are expected according to saturation hybridization experiments (Hattlen & Attardi, 1971). The known examples demonstrate that individual members of tRNA gene families are not reiterated, but rather dispersed throughout the human genome, with most of them occurring alone on quite large restriction fragments (Santos & Zasloff, 1981; Doering et al., 1982; Arnold et al., 1986; van To1 & Beier, 1988). There are also examples for clustered tRNA genes and pseudogenes for the same tRNA species (Sekiya et al., 1982; Morry & Harding, 1986; Pirtle et al., 1986; Doran et al., 1987) and for clustering of genes and pseudogenes of different tRNA species (Roy et al., 1982; Looney & Harding, 1983; Makowski et al., 1983; Shibuya et al., 1985; MacPherson & Roy, 1986). Of the nine characterized human tRNA”“’ genes, five (i.e. those encoded by pHtV2, pHtV3, pHtV4, pHtV5 and pHtV8) are apparently the only tRNA genes within the surrounding sequences examined by sequencing and hybridization analyses. However, the cloned fragments in case of pHtV3 (Arnold et al., 1986) and pHtV4 (Kahnt et al., 1989) are quite small and we cannot exclude the possibility that there might be more tRNA genes in their neighbourhood beyond the EcoRI sites. For clones pHtV1 and pHtV6, subfragments of the initial lambda clones hybridized against labelled total human tRNA. However, they did not give rise to transcripts in vitro and therefore presumably are pseudogenes. (b) Is there an evolutionary tRNA”“’

Gene duplication account

relationship genes?

between

events have been proposed to

for the dispersion

of tRNA

genes (Santos

&

Zasloff, 1981). Evidence for gene duplication can be obtained by comparison of sequences of flanking

A Human

- ; c

517

Gene Family

tRNA

;m.<.... t :-CD

a&

U---L K:-‘”

i--c c--c G--C

60 ‘p,” CCCCCi’. 11 c “b CCCCG”“C , ‘C c u-4, CY GG-16 U-.

c--c “G’” c G U”.“-“-&

CUG I !

“-4 c--c c--c C--G

c--c 30-c - c-‘o cc--=c ” 4 ‘.C

(,j)

:

CG:CC”. , CCYCG:“C

,,

c .

‘.C

-L: CU.“““” -1 :“(e) & ;-a .t t.

-‘ < - ‘ L”““” -i “ti-4 “-4 “-. Y-. t--G c--6 G-“-i

. 9;r~“‘, cccGG”“c c;

YC’“Cyt’U c t ““.“C&“-.. “-“C C--t G--C C--t :

l c c ’ ” G UC c I ’ c ““A UC ‘C.

.“=-c,

C.C

c .

C--t

,”

l

ccc , ccc =u

cc:;. I = CC”“C

t--G c--c CA’. I c C”“C

“-.IcG “-.

,” UC’” 6°C t / / I G ““.“C’CC”-&

c--c c--c c--c

.A c

c :

”

. C’C

c-i

CCCCCA’. IIIII ccccc”“:

“- L c-c c -,’

r

=.c

-.

Figure 7. Cloverleaf structures of 9 pre-tRNA”“’ species. The information about primary and secondary structures of pre-tRNAs derived from the 9 tRNAVa’ genes is summarized. Only those primary transcripts derived from pHtV1 to pHtV9 (a) to (i) are shown that were terminated at termination site(s) located next to the 3’ ends of the structural genes. i.e. long readthrough products are not shown. Major and minor transcription initiation nucleotides are labelled by large and small arrowheads, respectively. The underlined sequences indicate heterogenous 3’-OH ends of pre-tRNAs due to transcription termination within the termination signals downstream from the tRNA genes. Primary sequences that are specific for the mature tRNA domains are shown with enlarged letters. Arrows and corresponding encircled numbers indicate processing sites and the determined sequence of processing reactions, respectively. In case of pHtV2-derived pretRNAs shown in (b). the correct 3’ processing site could not be determined exactly, and 2 possible cleavage sites are indicated by arrows and a question mark. Base changes in the mature tRNA domains compared with pHtVl-encoded tRNA are marked by dots.

regions of genes stemming from a common ancestor. For instance, human tRNAArg genes show remark-

able conservation in their neighbouring sequences (Ma et al., 1984). In the case of the tRNA”“’ gene family, we found only one example where a close relationship between the sequences adjacent to the structural genes could be detected, i.e. between pHtV3 and pHtV6 (Fig. 2). From the amount of mutations that have accumulated in their flanking sequences, the time-span that has elapsed since a putative duplication event can be calculated. Using the methods described by Kimura (1977) and modified by van Ooyen et al. (1979), we obtained a value

of about 40 million years (30 to 50 million years using different values for k,,, or for the degree of similarity between aligned random sequences), which means that the genes developed from a common ancestor well after the mammalian radiation (65 million years ago) but before the separation of humans from the other primates. (c) Alu repetitive

elements

Six of the nine known repetitive

bourhood.

element

of the Ah

next to tRNA’“’

tRNA”“’ family

This is also common

genes

genes have a in their

neigh-

for other human

518

H.-U.

Thomann et al.

tRNA genes (Buckland et al., 1983; O’Neill et al., 1985; Shortridge et al., 1985; Pirtle et al., 1986; Doran et al., 1987). Statistical calculations suggest a slight over-representation in the surroundings of the nine tRNA”“’ genes (one AZu element within about 2 kb of flanking sequences as compared to 1 Alu member within 5 kb of genomic DNA in general) as far as sequence data are available. One explanation for this over-representation is perhaps the A+Trich flanking sequences of tRNA genes that Alu elements are known to prefer as integration sites (Daniels & Deininger, 1985). Although it has been reported that polymerase III promoter boxes, including those of AZu elements, can enhance the transcription of downstream polymerase II genes (Oliviero & Monaci, 1988), there is no evidence for an influence of Alu sequences at least on expression of tRNAVa’ genes in vitro in any case that has been investigated so far.

(d) The tRNA”“’ gene family: genes, variant genes and pseudogenes Of the nine different tRNAVa’ genes, only four code for one of the known mammalian tRNA”“’ isoacce tors, i.e. three for tRNA,V,“:, and one for tRNA,&J If one regards our collection of genes that covers more than half of the human tRNAVa’ genes (Arnold et aE., 1986) as representative, there is a discrepancy between the number of genes coding for each of the two isoacceptors and their concentration in human tissue, provided that this concentration is determined by gene dosage as it has been demonstrated for D. melanogaster tRNAVa’ genes (Larsen et al., 1982). In placenta, the ratio of tRNAEA to tRNAEfc is 2 : 1 (Chen & Roe, 1977) as compared to 3 : 1, which is the ratio of the corresponding genes. This discrepancy is further exaggerated by the very low transcriptional activity in vitro of the gene encoded by pHtV3. It is possible that there may be other yet undetected tRNA$$ genes with especially high transcri tional activities. Alternatively, not all of the tRNA&$ genes may be active in vivo. It should be noted that tRNA$ may not be required absolutely in all tissues, since tRNA,V,“:, itself recognizes all four valine codons, at least in vitro (Jank et al., 1977b). Although corresponding tRNAs derived from clones pHtV5 and pHtV7 (Figs 1, and 7(e) and (g)) have not been identified in vivo, we do not want to exclude the possibility that these genes are functional and are coding for minor isoacceptor species. It is known that a tRNAVa’ species is present in human placenta that has not been characterized so far (Anandaraj & Roe, 1975). At least one of these variant genes may therefore be an “allogene” rather than a “pseudogene”. This would be in agreement with the variant tRNAVa’ genes found in D. melanogaster (Addison et al., 1982; Larsen et al., 1982; Leung et al., 1984).

(e) Are there extragenic control elements in the jlanking sequences of the tRNA”“’ genes? Transcription of tRNA genes is directed primarily by a bipartite intragenic promoter that spans residues coding for the DHU arm (nucleotides 8 to 19, the so-called A-box) and the TYC arm (nucleotides 52 to 62, the B-box: Galli et al., 1981; Hofstetter et al., 1981; Sharp et d., 1981a). Yet there is no doubt that sequences outside of the structural gene also have a substantial influence on transcription of tRNA genes in vitro and in vivo (for reviews, see Sharp et aE., 1985; Geiduschek & Tocchini-Valentini, 1988). Most investigations localize the sequences responsible for this effect between nucleotides - 1 to about -70 of the 5’ flanks, although the 3’ flanks up to about 50 nucleotides downstream from tRNA genes, apart from the common oligo(T) transcription termination signal, can also contribute to extragenic modulation (Schaack et al.. 1984; Wilson et al.. 1985; Lofquist & Sharp, 1986; Raymond & Johnson, 1987; Arnold et al., 1988). There is only limited information about extragenic control elements of mammalian tRNA genes. For instance, transcription and stable complex formation of murine tRNA”‘” genes are influenced by a conserved leader sequence (Morry & Harding, 1986) and transcription of the human tRNAVa’ gene encoded by pHtV1 exhibits a flanking sequence dependence, which suggests the presence of a positively acting control element (Arnold & Gross, 1987). However, conserved sequence motifs that reflect functional requirements concerning flanking regions of the tRNA”“’ genes and represent consensus sequences for extragenic transcriptional control were not found. Moreover, no consensus could be detected by comparing flanks of the tRNA”“’ genes with those of the other known human tRNA genes. Although they lack consensus sequences, the flanks nevertheless are responsible for the different transcription efficiencies of the three tRNA$$ genes encoded by pHtV1, pHtV6 and pHtV8, as the structural genes are identical. Arnold et al. (1986) demonstrated previously that exclusively flanking regions cause the low transcriptional activity of the tRNAz$ gene of pHtV3. This structural gene is identical with those mentioned above, except for the anticodon which, however, was shown to have no impact on transcription. A point to which only little attention has been addressed so far (Zasloff et al., 1982c) is the magnesium ion dependence of tRNA gene transcription. We found that the transcriptional activity of most of the cloned tRNAVa’ genes exhibits a quite sharp Mg2+ optimum, whereas one of them, i.e. pHtV1 is remarkably active over a comparatively wide concentration range and reaches its maximal efficiency slightly later than the other genes. The different Mg ‘+ dependencies of the tRNAVa’ genes encoded in pHtV1, pHtV3, pHtV6 and pHtV8 suggest that, like the different transcriptional activities in vitro, this effect is caused by the flanking sequences.

A Human tRNA Gene Family Altogether, no general model for transcriptional control directed by flanking re ions can be designed so far, neither for the tRNA”” Qgene family, nor for human tRNA genes in general. Conclusively, we think that any attempt to get an insight into extragenie transcription control of tRNA genes by searching for sequence homology alone may be too simplistic. Quite different sequences can serve the same function, as for instance melting of DNA strands to facilitate access of the coding strand for factors and/or the synthesizin polymerase. Especially the 5’ flanks of tRNAVa F genes often contain stretches of poly(A) and poly(T), respectively.

(Q Transcription initiation at human tRNA”“’ genes in a homologous nuclear extract Polymerase III is known to initiate transcription preferentially with a purine located near the 5’ end of a tRNA gene. Therefore, pre-tRNAs contain purine nucleoside triphosphates at their 5’ ends (De Robertis & Olson, 1979). However, the selection of initiation site(s) does not seem to follow a simple rule, neither with respect to tRNA gene families nor to transcription systems. There are many examples for the selection of only one initiation site in the 5’-flanking sequence of tRNA genes (Silverman et al., 1979; Cooley et al., 1984; Rooney & Harding, 1986; Mazabraud et al., 1987; Vnencak-Jones et al., 1987; Ganguly et al., 1988) and some cases with two or more sites that are used equally (Hofstetter et al., 1981; Sharp et al., 1981b; Koski & Clarkson, 1982; Hipskind & Clarkson, 1983). The impression that mainly a single site is available for transcription initiation may be incorrect, since fingerprint analyses are in general not sensitive enough to detect oligonucleotides that derive from minor transcription products. Moreover, the 5’-terminal triphosphate residue that identifies the first nucleoside of a transcript is often labile, depending on the transcription extract used. For these reasons we preferred to use reverse transcription of pre-tRNAs in order to map their 5’ end and to detect even minor transcription initiation sites. As mentioned in Results, the whole pre-tRNA population obtained by transcription of any of the tRNAVal genes was analysed accordingly. All initiation sites found for each of the nine tRNAVa’ genes are indicated in Figure 7 and, as expected, the fingerprints revealed only the major initiation sites. Interestingly, there is no example for a pre-tRNA that is initiated at one site only. In general, there are two to four initiation sites for each tRNA gene. A rule for the selection of those sites obviously does not exist. They are spread within the range of -2 to -9, with a preference for -4 and -6. Of the nine tRNA”“’ genes, eight initiate transcription at - 4 and/or - 6. In contrast, the other positions are less frequently used. Transcription initiation of tRNA genes in HeLa cell and Xenopus kidney cell extracts seems to be similar in some reported cases, i.e. with a slight preference at -4 and - 6 (Koski & Clarkson, 1982; Hipskind &

519

Clarkson, 1983; Vnencak-Jones et al.. 1987; Ganguly et al., 1988). In accordance with the feature that initiation starts preferentially at purine bases that frequently are 3’ neighbours of a pyrimidine base (for a review, see Clarkson, 1983), we found that 16 of 25 identified initiation sites in front of the nine human tRNAVa’ genes follow that rule. However, there are two exceptions; namely, the initiation sites at C ( - 2) and C ( - 6) of the tRNA genes of pHtV3 and pHtV7, respectively. The apparent initiation at C (-2) (pHtV3) is more likely to be due to fast degradation of pre-tRNA as reported by Zasloff et al. (1982a), since the intensity of the corresponding cDNA band (Fig. 5, lane 3) correlates with that of a major initiation site, but the fingerprint analyses did not reveal any indication for this initiation site. In contrast, the initiation site at C (-6) (pHtV7) was proven by reverse transcription (Fig. 5, lane 7) and fingerprint analyses (Fig. 4 A7). A fast degradation of precursors seems very unlikely, as the corresponding cDNA band is the longest product obtained by reverse transcription of pHtV7-derived pretRNA. To our knowledge, this would be the first observed case of a polymerase III transcription initiation with a pyrimidine base in vitro.

(g) Transcription termination their eficiency

signals and

The first indications that polymerase III terminates transcription of tRNA genes within a short cluster of thymidine bases located downstream from the structural gene arose when pre-tRNAs were found to contain heterogenous 3’ ends that differ only by the amount of uridine bases (Garber & Gage, 1979; Hagenbiichle et al., 1979; Silverman et al., 1979). Hofstetter et al. (1981) and Galli et al. (1981) confirmed the notion that short stretches of thymidine bases downstream from tRNA genes serve as transcription termination signals, whereas Bogenhagen et al. (1980) had shown that this is true also for 5 S rRNA gene transcription termination. Today, it is widely accepted that at least a tetrathymidine stretch surrounded by G and C residues is sufficient for polymerase III termination, although cases diverging from this rule are still increasing (for reviews, see Clarkson, 1983; Sharp et al., 1985; Geiduschek & Tocchini-Valentini, 1988). Sharp et al. (1981a) reported that TTCTT instead of TTTTTTT in the same location after a tRNA gene serves as a stop signal, albeit with lower efficiency, depending on the transcription extracts used (Schaack et al., 1984). A similar sequence is responsible for transcription termination downstream from the tRNA genes of pHtV2 and pHtV6. In the first case, termination occurs at CTTCTT 11 bp downstream and at GTCTT 19 bp downstream from the tRNA gene. The efficiencies are high; about 70% of the transcripts are terminated within these two sequences, whereby the first signal seems to act more strongly (Fig. 3(b), RNA 1 and II). The rest of

520

H.-U.

Thomunn

the transcripts terminate farther away at conventional termination signals (see Results). The effect of the ATCTT sequence 3 bp downstream from the tRNA gene of pHtV6 is much more striking than the similar sequence (GTCTT) 19 bp downstream from the pHtV2 DNA. Nearly 90% of all transcripts are terminated within these nucleotides. Important factors influencing termination efficiency are probably the nucleotides surrounding the oligothymidine stretches. Mazabraud et al. (1987) showed that CTTTTA beyond a Xenopus tRNALyS gene is a considerably stronger terminator than CTTTTC. The latter is identical with the termination sequence of pHtV3 tDNA and facilitates similar readthrough transcription. The weakly acting termination signals of pHtV7 and pHtV8 tDNA (Figs I, and 3(f) and (g); 30% and 20% readthrough transcription, respectively) are each surrounded by G and C residues. In contrast, the efficient terminator of pHtV5 tDNA (Figs 1 and 3(d)) is flanked by A and C (ATTTTC), like that of the Xenopus tRNALyS gene, though in the opposite order. The strongly terminating TCTT sequence located beyond the structural gene of pHtV6 is even flanked by two adenosine bases (ATCTTA). Therefore, in contrast to 5 S rRNA gene transcription termination (Bogenhagen et al., 1980; Bogenhagen & Brown, 1981), we propose that at least’ the termination signals of human tRNAVa’ genes are strengthened by surrounding A residues. Recent studies of a new unusual terminator (ATATATATT) after polymerase III-transcribed salmon-derived pal/SINE sequences showed that its efficiency also appears to be dependent on its location relative to the transcription unit and its neighbourhood (Matsumoto et al., 1989). Hess et al. (1985) observed that transcription termination of a polymerase ITTtranscribed human Alu repeat occurred at a stretch of nine adenosine bases approximately 255 bp downstream from the initiation site. The efficiency depended on the presence of immediately 3’-adjacent A + T-rich sequences. Finally, we have observed that transcription termination is influenced by Mg2+; the amount of readthrough products increases at concentrations above 3~5 mM-Mg 2+. Furthermore, termination efficiency and the amounts of readthrough products in the cases of pHtV2, pHtV3 and pHtV6 to pHtV8 transcription are strongly dependent on the extract preparation. (h) The 5’ and 3’ end maturation pre-tRNAVn’

of

species

It has been shown that eukaryotic pre-tRNAs are restricted to the nucleus, and that the processing, i.e. cleavage of leader and trailer sequences of a pretRNA, creates mature tRNA that only then can penetrate the nuclear membrane and enter the cytoplasm (Melton & Cortese, 1979; Melton et al., 1980; Zasloff et al., 1982h; Zasloff, 1983; Tobian et al., 1985). It is assumed that maturation of pre-tRNAs and active release of their products from one

et al.

compartment to the other represents a kind of posttranscriptional control mechanism that excludes defective tRNAs (Zasloff et aZ., 1982a). We show here that transcripts derived from pHtV4 and pHtV9 are not processed at all, and hence may not leave the nucleus if they also occur in vivo. The pretRNAs of six genes (pHtV1, pHtV3, pHtV5, pHtV6, pHtV7 and pHtV8) are accurately processed to 73 nucleotide long mature tRNAsVa’ that would be able to enter the cytoplasm after the 3’-terminal CCA sequence has been added (Melton et al., 1980). The pre-tRNA derived from pHtV2, however, is processed incorrectly and does not yield a mature tRNA”” (see Results). (i) The processing-defective

pre-tRNAs

The effect of the mutations in the tRNA gene of pHtV4 and their reversions has been discussed extensively by Kahnt et al. (1989). As shown in Figure 7(i), pHtV9-derived primary transcripts do not form a normal anticodon stem due to an A *C mismatch. This mutant pre-tRNAVa’ is neither a substrate for the 3’ nor for the 5’-processing enzymes. The processing defect of this precursor accords with previously described examples (Nishikura et al., 1982; Harada et aZ., 1984; Pearson et al.. 1985; Willis et al., 1986). (ii) The accurately processed pre-tRNAs The primary transcripts derived from each of the tDNAs of pHtV1, pHtV3, pHtV5, pHtV6, pHtV7 and pHtV8 are accurately maturated by removal of the 3’ trailer and 5’ leader sequences and addition of the 3’-terminal CCA sequence. As shown in Results, processing occurs first at the 3’ ends and subsequently at the 5’ ends, since no 5’-processed intermediates with trailer sequences could be detected. The same sequence of maturation reactions seemed to occur during processing of a yeast pre-tRNATY’ (De R’obertis &, Olson, 1979) and a Paenorhabditis elegans pre-tRNAL”” in Xenopus oocytes (Melton & Cortese, 1979). Gruissem et al. (1982) showed that polycistronic E. co&i and Euglena chloroplast pretRNAs were processed primarily at the 3’ ends in a HeLa cell extract. Hagenbiichle et al. (1979) and Garber & Gage (1979) also observed the cleaved trailer of Bvmbyr pre-tRNA*‘” created by the action of a 3’ endonuclease in a Xenopus germinal vesicle extract. It is rather likely that a 3’ endonuclease is also responsible for 3’ maturation of the pre-tRNAs”“’ described here. since we never observed stepwise degradation products expected for a 3’ to 5’ exonucleolytic removal of trailer sequences. In the case of an altered sequence of maturation, i.e. 5’ prior to 3’ processing, a 3’ endonuclease was found in a Drosoph,ila cell-free extract (Frendewey et aZ., 1985) and in an extract from Xenopus ovaries (Castano et al., 1985). However, a 3’ exonuclease activity has been identified in a Xaccharvmyces cerevisiae extract (Engelke et al.. 1985). At this point, it should be noted that the sequence of the processing events is the same for long readthrough transcripts obtained upon t’ran-

A Human

tRNA

scription of pHtV3, pHtV7 and pHtV8 (Fig. 3(c), (f) and (g)) as for the transcripts that terminate closer to the 3’ ends of the genes (see Results). These findings are in accordance with results reported by Galli et al. (1981), Hofstetter et al. (1981) and Adeniyi-Jones et al. (1984), but in a certain contrast to those of Allison & Hall (1985). By-products of the 3’-processing reactions that are detected during maturation of pHtV3, pHtV7 and pHtV8-derived pre-tRNAs are partially 3’-degraded precursors. In all cases, a nuclease produces precursors with a four to six nucleotides long, truncated trailer. We do not know whether these cleavage products are created by the same nuclease that is normally involved in 3’ processing. Activities cleaving pre-tRNAs some residues downstream from the mature domain were detected in prokaryotes (Ray & Apirion, 1981; Nomura & Ishikama, 1988; for a review, see Altman et al.. 1982). mentioned, 5’ processing of the As alread 9 occurred in all cases after 3’ maturapre-tRNAs”” tion. It remains unknown whether an RNase P-like activity that has been detected in HeLa cells (Gold & Altmann, 1986) is responsible for the 5’ maturation of the pre-tRNA”“’ species. Interestingly, 3’.processed pre-tRNAs, especially those with mature 3’ CGA sequences, have been shown to be better substrates for RNase P in prokaryotes and eukaryotes (Schmidt et al., 1976; M&lain et al..

1987). Finally, it should be mentioned that the variant species obtained by transcription of pre-tRNAVa’ the genes of pHtV5 and pHtV7 are accurately mat)urated to tRNAs. It appears that these precursors are as good substrates for the processing enzymes as those derived from pHtV1, pHtV3, pHtV6 and pHtV8. The major tRNAVa’ has been shown to contain an additional U54. A60 base-pair in the Tr,!K! stem (Jank et al., 1977a). This base-pair is absent in pHtV7-derived RNA (Fig. 7(g)). Even this precursor is correctly processed, indicating that the processing activities are not dependent on this extra base-pair of the T-loop. The sequences of the leaders and trailers of the different precursors do not dramatically influence the velocit’y of processing, although pHtV6-derived transcripts seem to be maturated more rapidly than those of the other genes. (iii)

A II incorwctly processed pre-tRNA”“’ It appears that both the shorter and the longer pHtV2-derived pre-tRNAs (Fig. 3(b)) are first cleaved in their 3’ trailer sequences. In contrast to the precursors derived from pHtV1, pHtV3 and pHtV5 to pHtV8 (Fig. 3(a), (c) and (d) to (g)), this cleavage occurs not exactly between the mature tRNA domain and the 3’ trailer. It is possible that the same act(ivity that produces the partially 3’processed intermediates in the case of the pretRNAs derived from pHtV3, pHtV7 and pHtV8 (see above) is responsible. In spite of the partially mat,urated 3’ end, the intermediate of pHtV2derived pre-t,RNA seems to be a substrate for the 5’

521

Gene Family

processing activity, since the accumulating product (Fig. 3(b), RNA V) contains the 5’ end of mature tRNA”“‘. The tRNA”“’ gene of pHtV2 has four mutations (T51, A54, A57 and A63) as compared with that of pHtV1 (Arnold et al., 1986). These changes do not affect base-pairs that alter the overall shape of the precursors, or the known consensus sequence of the B-box domain (Galli et al., 1981; Hofstetter et al., 1981; Sharp et al., 1981a). The T to A transversion at position 54 destroys the tRNA”“‘-specific, sixth base-pair in the Tt& stem, but this should not affect the processing events of this pre-tRNA, since this is absent also in pHtV7derived precursors, which are correctly maturated to tRNA”“’ (see above). Traboni et al. (1984) have shown that a T54 to A54 mutation in a tRNA gene did not affect the processing of its precursors. Finally, the U51. A63 base-pair occurs also in other eukaryotic nuclear tRNAs, especially in initiator tRNAMe’, tRNAAs” and tRNAS”’ (Sprinzl et al., 1989). For these reasons, we assume that the mutations in the tRNA gene of pHtV2 are not responsible for incorrect processing of its precursors. The abnormal maturation events of this pre-tRNA are more likely caused by leader and trailer sequences that are able to form secondary structures resulting in an extended aminoacyl stem. The U residues of the leader at positions - 1 and - 2 can base-pair with the last A of the tRNA domain and the first A of the trailer sequence (Fig. 7(b)). The extended aminoacyl stem of this precursor may be responsible for the reduced or abolished recognition of the correct 3’ maturation sit#e by the respective processing enzyme. The trailer is cleaved only three to four residues downstream from the mature tRNA domain. It has been shown that tRNA precursors containing such an extended aminoacyl stem were processed correctly ,i,n viva, although with reduced velocity; however, precursors with mismatches within the 5’ and 3’ cleavage sites were processed in a normal manner (Castagnoli et al., 1982). Finally, we assume that the incorrectly 3’. processed pre-tRNA”“’ is no longer a substrate for the 3’-processing activity as soon as it is cleaved by the 5’-maturating enzyme, since this product is not) processed to mature tRNA in HeLa cell extract,. LYe thank I’etra Steinmiiller and I’et,er Scharff for assistance in cloning and sequencing. and Dr H. Reier for reading the manuscript. This work was supported hv Sondrrforschungsbereich 165 and by Fonds der (Ihemwhen

Industrir.

References Addison. W. R.; Astell, C. R.. Delaney, A. D., C:illam. T. C., Hayashi, S., Miller, R. C., Rajput. B.. Smith, M., Taylor. D. M. & Tener. (2. M. (1982). .I. Bid. Chem. 257, 670-673. Adeniyi-Jones, S., Romeo, 1’. H. & Zasloff, M. (1984). Nucl. Acids Res. 12, 1101-1115. Allison, D. S. & Hall, B. I). (1985). EMBO J. 4, 26.57. 2664. Altman, 8.. Guerrier-Takada, C.. Frankfort. H. M. &

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Chen, E. Y. & Roe, B. A. (1977). Biochem. Commun. 78, 631-640. Clarkson, S. G. (1983). In Eukaryotic Structure,

Activity

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Biophys. Genes:

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