Proc. NatL Acad. Sci. USA Vol. 79, pp. 1921-1925, March 1982 Genetics

Relationship between the two components of the split of eukaryotic tRNA genes (hybrid gene/insertion mutants/nuclear microinjection/RNA polymerase HI ) G. CILIBERTO*, C. TRABONI*, AND R. CORTESE European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, Federal Republic of Germany Communicated by Sydney Brenner, October 21, 1981

ABSTRACT Plasmids containing eukaryotic tRNA genes are stitute the split promoter of a tDNAPro from Caenorhabditis faithfully transcribed in the nucleus of Xenopus loevis oocytes elegans (6) and of a tDNAMet from Xenopus laevis (5). [Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) In the light of this idea one can recognize at least two other Nucleic Acids Res. 5, 4593-4611]. It has been established that two structural features that must be common to all tRNA genes. One separated regions within the coding sequence of a tRNA gene are is the physical distance between the two invariant regions: this essential and sufficient for promotion oftranscription [Hofstetter, is about 40 nucleotides in almost all tRNAs, with the exception H., Kressmann, A. & Birnstiel, M. L. (1981) Cell 24, 573-585; of those tRNA genes that contain . The other common Ciliberto, G., Castagnoli, L., Melton, D. A. & Cortese, R. (1982) feature is the Proc. Natl. Acad. Sci. USA 79, 1195-1199]. We have constructed overall partial self-complementarity ofthe 5' and a hybrid tRNA gene containing one essential region from tDNAIeU 3' halves ofa tRNA molecule, which confers on the correspond- and the other from tDNAPrO, both from Caenorhabditis elegans. ing DNA sequence the potential for folding into a cloverleaf-like This hybrid gene is efficiently transcribed, thus showing that the secondary structure. We asked ourselves ifany ofthese features essential regions are independent transcriptional signals regard- play a role in transcription. less of the overall regularities of the structure oftRNA genes. We In order to provide an answer to these questions we have have also constructed mutants of the tRNA"' gene in which the constructed two different sets of mutant tRNA genes. In one distance between the two essential regions is changed; optimal set we varied the distance between the two essential regions; transcription occurs when this distance is about40-50 nucleotides. in the other we recombined "essential regions" so as to obtain a new "gene" whose promoter is composed ofa 5' halfderiving It has been firmly established that the genes transcribed by from tDNALu and the 3' half from tDNAPr, from C. elegans RNA polymerase III contain their transcriptional signals (pro- (tDNA is the DNA coding for tRNA). moters) within the coding sequence. In 5S RNA genes a 30-base pair (bp) region seems to contain all the information necessary EXPERIMENTAL PROCEDURES for correct initiation oftranscription (1, 2). In the VAI RNA gene Recombinant DNA work was performed according to the rec- of adenovirus an internal control region, approximately 60 bp ommendations outlined in the National Institutes of Health long, could be identified (3, 4). In the case oftRNA genes a more guidelines ofJune 23, 1976. precise analysis leads to the identification of two sequences of Purification of Flush-End DNA Fragments from Plasmid about 10 nucleotides each, located within the , pBR322. Double-stranded pBR322 DNA was cut either with whose presence is essential for transcription (5, 6). This overlap restriction endonuclease Alu I (Boehringer) or with restriction ofcoding sequences and transcriptional signals is characteristic endonuclease Hae III (gift ofV. Pirrotta). DNA restriction frag- of eukaryotic organisms: the corresponding genes (5S RNA, ments were separated electrophoretically on Tris glycine gels tRNA) in prokaryotes have aclassical promoter clearly separated (11). The section of the gel containing fragments 19 to 201 bp from the coding region and located in the 5' flanking region long was cut out and the fragments were eluted as described (7). Because the structure oftRNA is very similar in eukaryotes elsewhere (12). and prokaryotes (8), the obvious conclusion is that the DNA Construction of Insertion Mutants. Plasmid pBcetl3 was coding sequences have not been modified to be adapted to the first cut with the restriction endonuclease Sma I (purchased new function of transcriptional signals, but rather the opposite from Boehringer) and then ligated in the presence ofeach ofthe must have occurred, namely that the transcriptional machinery previously purified flush-end DNA fragments from pBR322. Phage T4 DNA ligase was a gift of V. Pirrotta. has evolved the capacity to recognize parts of the coding se- Escherichia coli K-12, strain Hb 101, was used for transfor- quence as transcriptional signals. Because all tRNA genes are mation. Protocols for transformation and preparation ofdouble- transcribed by RNA polymerase III (9) it is reasonable to expect stranded DNA were as described (11). Transformants grown on that the transcriptional signals must correspond to features com- LB plates supplemented with ampicillin at 100 Ag/ml were mon to all tRNA genes. One of these features is the so-called screened for the presence of the inserted fragment. This was invariant nucleotides. On the basis of the analysis of several done by rapidly preparing plasmids on a microscale (13) and mutants of a tRNATYr gene of yeast Koski et al. (10) have hy- then lookingfor the loss ofthe Sma I recognition site. The length pothesized that the internal promoter of a tRNA gene is essen- of the insert was established by comparing the total length of tially constituted by these invariant nucleotides. It is interesting the coding region in the wild-type gene (plasmid pBcetl3) and that these invariant nucleotides are particularly, although not in the insertion mutants, after digestion with restriction en- exclusively, concentrated in the two essential regions that con- donucleases HindIII and BamHI (purchased from Bethesda

The publication costs ofthis article were defrayed in part by page charge Abbreviations: bp, base pair(s); tDNA, DNA coding for tRNA. payment. This article must therefore be hereby marked "advertise- * On leave of absence from Istituto di Chimica Biologica, II Facolta di ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Medicina, University of Naples, Naples, Italy. 1921 Downloaded by guest on September 28, 2021 1922 Genetics: Ciliberto et aL Proc. Natl. Acad. Sci. USA 79 (1982) Research Laboratories). DNA polymerase large fragment (Kle- now enzyme) used to end-label DNA fragments was purchased from Boehringer. Construction of a tDNAI"U-1)NAP' Recombinant Gene. p~~cetl3~~PBR322 The parental plasmids were Mcet7, containing a 250-bp nem- p~~cetl3~~cut withAlu "I atode DNA insert with a single copy of a tDNAJeU from C. \ / \ \ ~~~~~~~orHae HI / elegans and Mceti, containing a 263-bp nematode DNA insert /EcoRI with a single copy ofa tDNAP"". The vectorwas phage mp2 (14). tDNA\~\ tDNAF'ror~ _o All the steps needed for the construction ofthe hybrid gene are BamHI _ HindJII represented in Fig. 5. Recombinant DNA linkers were pur- Sma I chased from Collaborative Research, Waltham, MA. Endonu- Cut with Sma I Blunt-end ligation of pBcetl3 clease Hha I and S1 nuclease were purchased from Bethesda with purified fragments of Research Laboratories. pBR322. Transformation, Microinjection and Quantitation Analysis. Microinjections purification of cloned DNA, into the nuclei ofXenopus oocytes were performed as described digestion with BamHI and (11). All plasmid were injected at the concentration of Hindll terminal labeling. 250 ,tg/ml. RNA transcripts were separated electrophoretically with 32 on 5% or 8% polyacrylamide gels run in TBE buffer (11). Quan- titative analysis was performed as follows: Bands were cut and 1 2 3 4 5 G T 8 9 10 ii _ . __ their radioactivities were measured as Cerenkov counts. A nor- ON. - __d - malization was then done, using as internal standard the radio- 4 activity incorporated-in the endogenous 18- and 28S rRNA spe- cies. This was normally done by a previous fractionation of a separate aliquot on a 2% agarose gel followed by measurement 4 of the radioactivity contained in the gel slices. In practice we 4 found that identical values were obtained also when, rather than 4i4 fractionating the rRNA on a separate gel, we normalized to the b radioactivity present at the top ofthe polyacrylamide gels. We I chose 18- and 28S rRNA as internal standard rather than 5S RNA because in this smaller molecule there was little incorporation 4 I ofradioactive precursors. We discarded the alternative ofcoin- jecting a purified 5S RNA gene, as was done by Hofstetter et al. (5), because we found that there is a considerable degree of -45>5iInr s~ competition with tRNA genes (15). FIG. 2. (Upper) Construction of insertion mutants of tDNA~'. Radioactive Compounds and Autoradiography. All radio- (Lower) Ten percent polyacrylamide gel electrophoresis of the end-la- active compounds were purchased from Amersham Buchler, beledHindl/BamHI DNA segments from the wild-type gene and the Braunschweig. Fuji films were used, occasionally with preflash- various insertion mutants: lanes 1 and 11, end-labeled Hinfl digest of ing according to Laskey and Mills (16). pBR322 as size marker; lane 2, pBcetl3 (wild-type gene); lane 3, pBcetl3/1; lane 4, pBcetl3/2; lane 5, pBcetl3/3; lane 6, pBcetl3/4; lane 7, pBcetl3/5; lane 8, pBcetl3/6; lane 9, pBcetl3/7; lane 10, RESULTS pBcetl3/8. Mutants carry the following segments from pBR322 (17): pBcetl3/1, 2117-2135; pBcetl3/2, 2068-2116; pBcetl3/3, 3656-3718; Construction of Insertion Mutants of a tRNAPrO Gene. In pBcetl3/4, aduplication offiagment 2068-2116, pBcetl3/5, 3556-3655; a previous study of the structure of the promoter of a tRNAPrO pBcetl3/6, 2643-2778; pBcetl3/7, 1262-1445; pBcetl3/8, 3556-3756. gene from C. elegans we have defined three regions important for the promotion of transcription (6) (see also sequence re- (boundary ofthe b-c regions). We used as starting materialplas- ported in Fig. 1). Region a, contained between nucleotides mid pBcetl3 (Fig. 2), in which the nematode tRNA sequences +9 and +18, and region c, contained between nucleotides are cloned in pBR322 between the EcoRI and the BamHI sites, +50 and +60, which are essential: all the alterations we have with a HindIII linker separating the coding region from the 3' been able to introduce in these two regions cause reduction of flanking sequence. The tRNAPrO gene inserted in this clone has transcription to undetectable levels. The region between po- a deletion of the 5' flanking sequence; however, this deletion sitions +26 and +49 is less important: complete deletion ofthis does not affect the transcriptional activity of the gene (6). The region or mutation of certain nucleotides leads to decreased construction of insertion mutants (illustrated in Fig. 2) utilized efficiency of transcription but transcripts are still detectable. a unique Sin I site between nucleotides C-49 and G-50 of the To establish whether the physical distance between these two tRNA coding sequence. regions is important and to gain further insight into the prop- DNA fragments to be inserted in the tRNA gene were iso- erties ofthe RNA polymerase III transcriptional machinery, we lated from plasmid pBR322. We digested pBR322 with the constructed a series of mutants of the tRNAPrO gene carrying flush-end cutting enzymes Alu I or Hae III, and some of the inserts of various length between nucleotides C49 and G-50 fragments between 19 and 201 bp long were then individually ligated to Sma I-cut plasmid pBcetl3; the resulting recombinant 1 a 20 b 40 c 60 plasmids were characterized by the increased size of their TDNAPRO i HindIII/BamHI fragment (the whole coding region). This is shown in Fig. 2. TALEU -Sa Transcriptional Analysis ofInsertion Mutants. All the clones constructed were injected into the nucleus ofX. laevis oocytes FIG. 1. Coding sequences of tDNAMW and tDNALU from C. ele- gans. For tDNA', bracketed regions a and c are essential for to assay their capacity to function as templates for transcription. transcription. The results are shown in Fig. 3 A, A', and B. Downloaded by guest on September 28, 2021 Genetics: Ciliberto et al. Proc. Natl. Acad. Sci. USA 79 (1982) 1923 A A' B c

2 3 5 ' _3 1 2 3 4 5 6 7 8 5' 6' 8' ~ - wwwwwVP rY

z

_- 100 -a c

c EncogenoLs A4.I -S RNA

100 Lndogenois tRNA Increment in length of insert

Endogenous 5S RNA

FIG. 3. (A, A', and B) Polyacrylamide gel electrophoresis fractionation of the RNA transcribed in the nucleus of X. kaevis oocytes. (A) An 8% polyacrylamide gel: lane 1, pBcetl3; lane 2, Mcet20; lane 3, pBcetl3/1; lane 4, pBcetl3/2; lane 5, pBcetl3/3; lane 6, pBcetl3/4; lane 7, pBcetl3/ 5; lane 8, pBR322. Mcet20, carrying an insert of 10 bp (HindIII linker) between nucleotides C-49 and G-50, has been described elsewhere (6). This is an autoradiograph exposed for 5 hr. (A') Longer exposure (2 days) of the right part of the gel in A, to show (lanes 5', 6', and 7') the bands that are barely detectable in the short exposure (lanes 5, 6, and 7, respectively). In lane 8' are now evident the endogenous tRNA and the 5S RNA that was used as a molecular size marker, together with tRNAP' of lane 1 (A). (B) A 5% polyacrylamide gel: lane 1, pBcetl3/5; lane 2, pBcetl3/6; lane 3, pBcetl3/7; lane 4, pBcetl3/8; lane 5, pBR322. (C) Correlation between increment in length of insert and increment in size of transcript, both measured in nucleotides. Molecular weight markers were tRNAP"" (75 nucleotides) and 5S RNA (120 nucleotides). Arrows in A and A' point to the bands that were considered for these measurements. In every case we have considered only the major transcriptional product.

Fig. 3 shows that all plasmids carrying inserts between 10 and Efficiency falls ifthe two regions are closer together or further 100 bp do promote transcription of specific RNAs; genes car- apart. If this optimal relationship can be generalized to all eu- rying inserts longer than 100 bp are not detectably transcribed karyotic tRNA genes we would predict that, on average, tRNA (Fig. 3B). We could have detected transcription even at 1/10th genes carrying long introns should be transcribed less effi- ofthat obtained with plasmid pBcetl3/5, suggesting a threshold ciently than the others. effect when the two essential regions are separated by more Construction of a Hybrid tRNAI'u-tRNAPr' Gene. The sec- than 140 nucleotides. The transcribed RNAs are exactly the ondary structure ofall tRNA molecules requires that in all tRNA length expected if start and termination of transcription occur genes there must be inverted repeats. In order to investigate at the same sites as in the wild-type tRNA gene (pBcetl3). This the influence on transcription of the overall partial self-com- is best shown by the linear correlation between the increment plementarity of a tRNA gene we constructed a hybrid gene in size of the insert and the increment in size of the transcript (pBlp) carrying region a from tDNALeu (15) (of C. elegans) and (Fig. 3C). We have performed a two-dimensional chromato- region c from tDNAPrO, joined by a linker about 20 bp in length. graphic analysis (18) of RNase T1 digests ofthe transcripts from Mcet2O, pBcetl3/1, and pBcetl3/2 (data not shown). All of 100 _ them contain the trinucleotide CCG, which is uniquely coded by nucleotides in position 2, 3, and 4 ofthe coding region ofthe gene. even we tRNAPrO Therefore, though have not identified 0~~~ the exact initiation for every transcript, we can conclude that it must be located somewhere before base 2 of the coding region. =50 - These results indicate that the initiation and termination or points must be the same very close in all cases. We conclude o / I therefore that, although the presence of a c region in the tRNA gene is essential for promotion oftranscription, its distance from region a or from the initiation point does not influence the pre- wt +30 +60 +90 cise point where transcription starts. A estimate quantitative of Distance between regions a and c, in nucleotides the extent of transcription is shown in Fig. 4. In this figure we also plotted the extent of transcription obtained with two dele- FIG. 4. Correlation between efficiency of transcription and dis- tion mutants that actually shorten the distance between the tance between regions a and c of tDNAPrO. o, Values obtained with essential regions and that have been characterized elsewhere plasmids pBcetl9 and pBcet23 carrying 14- and 7-bp deletions, re- spectively, in the region between a and c; these plasmids are described (6). Transcription is most efficient when the distance is similar elsewhere (6). *, Insertion mutants described in this paper. wt, Dis- to that found in normal tRNA genes-i.e., 40-50 nucleotides. tance in the wild type. Downloaded by guest on September 28, 2021 1924 Genetics: Ciliberto et al. Proc. Natl. Acad. Sci. USA 79 (1982) A B

1 2 3 4 S

EcoRI

FIG. 5. (A) Construction of a hybrid tDNA~ k-ONA° gene. Mcet7 (18) carries a 250-bp Sau 3 segment inserted into the BamHI site of MpLL2 vector (19). Mcetl and pBcetl91 have been described (6, 12). The other represented plasmids were constructed in the following manner: pBcet74: Mcet7 DNA was digested with restriction endonuclease Hha I followed by S1 nuclease, to generate blunt ends. AfterEcoRI digestion the resulting DNA fragments were separated by 10% polyacrylamide gel electrophoresis (11). The 140-bp segmentEcoRI/HhaI containingthe 5' third of tDNAIMU was extracted, blunt-end ligated to Hindill linkers, digested extensively with EcoRI andHindm endonucleases, and inserted as theEcoRI/HindII fragment into pBR322 plasmid. p~cet45: pBcetl91 DNA was digested with EcoRI and HindEll endonucleases. The 175-bp EcoRI/Hindff segment containing the 3' third of tDNA was purified by polyacrylamide gel electrophoresis (11, 12) and cloned in pBR322 plasmid. pBlp: pBcet74 and pBcet45 DNAs were digested with Hindlu, ligated together, and subsequently cut with EcoRI. This procedure generated five low molecular weight DNA fragments. We fractionated these by 10% gel electrophoresis and purified the EcoRI/EcoRI fragment of 320 bp, corresponding to the hybrid gene. This was then inserted into pACYC184 plasmid (20) and cloned. (B) Fractionation by 10% polyacrylamide gel electrophoresis of RNA tran- scribed in the nucleus of X. Iaevis oocytes. Lane 1, Mcet7; lane 2, pBcetl91; lane 3, pBcet45; lane 4, pBcet74; lane 5, pBlp.

The starting plasmids were: Mcet7, carrying a 250-bp nematode a and c are essential, and their ability to promote transcription insert into the BamHI site ofphage mpLL2 (19) and coding for is optimal when they are separated by 40-50 nucleotides. In- a tRNALU (unpublished data), and Mcetl, coding for a tRNAP', creased proximity of these two sequences is inhibitory, as if which has been described (6, 12). The cloning strategy is rep- there were not enough space between them to allow correct resented diagrammatically in Fig. 5A. After microinjection into positioning of the various components of the RNA polymerase the nucleus of Xenopus oocytes, we observed that the hybrid III complex. On the other side of the curve of Fig. 4, that is, gene pBlp gave tRNA size transcripts with an efficiency com- when regions a and c are increasingly far apart, we observe a parable to one ofthe two parental plasmids (pBcetl91), whereas progressive decrease in the efficiency of transcription. pBcet74 and pBcet45, respectively carrying the region a of The residual transcription still detectable when the two es- Mcet7 and the region c of Mcetl alone, were not transcribed sential regions are separated by up to 140 nucleotides and the (Fig. 5B). Two-dimensional analysis (data not shown) of frag- absence of any detectable transcript when they are brought fur- ments of the pBlp transcripts reveals the presence of oligonu- ther apart deserves further comment. DNA of eukaryotic cells cleotides characteristic of the 5' half of tRNALeU (A-G, A-U-G, is packed into chromatin. There are, however, cases in which C-C-G, U-C-A-A-G) and ofthe 3' halfoftRNAPro (U-U-C-A-A- relatively long stretches of DNA appear to be naked (21). The U-C-C-C-C-G, U-U-C-G); apparently a hybrid RNA molecule interpretation of our results is necessarily different depending is transcribed from the hybrid gene. This result shows that re- on whether an actively transcribed tRNA gene is naked or gion a and region b can be active also within an artificial gene wound around nucleosomal particles. We know that the pres- that has lost the potential capacity to fold into a structure mir- ence ofregion c is essential for transcription, but that transcrip- roring that of a tRNA. In the tDNALeU-Pro (pBlp) the sequences tion starts at the 5' side of region a. The RNA polymerase III at its 5' and 3' ends are not complementary, like those found molecule from eukaryotic systems is considered to be a big in all natural tRNA genes. Also, the sequence coding for the molecule with many subunits (22); its size is probably large anticodon stem and loop is altered. Due to the presence of enough to be compatible with a hypothetical simultaneous bind- HindIII linkers there is in fact the potentiality to form a pseudo- ing with all the components ofthe split tDNA promoter. When anticodon stem, but the overall size of the anticodon region is the physical distance between regions a and c becomes far in reduced. excess ofthe dimensions one can reasonably assume for the RNA polymerase III molecule, a simultaneous interaction with the DISCUSSION two regions is impossible if the DNA is naked; in this case we On the basis of the results presented in this paper we can now must assume a multistage mechanism in which information trav- reevaluate the questions outlined in the Introduction. Regions els backwards from the end to the front of the gene. This could Downloaded by guest on September 28, 2021 Genetics: Ciliberto et d Proc. Natl. Acad. Sci. USA 79 (1982) 1925

tDNAPrO 8 NUCLEOTIDES - GGTCTAGTGG -- - 31 NUCLEOTIDES --- GGGTTCAATCC--12 NUCLEOTIDES the Xenopus oocyte. Below is a generalized sequence ofregions a and c ofall eukaryotic tRNA genes. The comparison shows that tDNALeu 8 NUCLEOTIDES- GGCCGAGCGG--- 41 NUCLEOTIDES --- GGGTTCGAATC--12 NUCLEOTIDES nucleotides 10, 14, 17, and 18 in region a and nucleotides 52, tDNAMe 8 NUCLEOTIDES - GGCGCAGCGG --- 31 NUCLEOTIDES --- TGGATCGAAAC--12 NUCLEOTlOES 54, 55, 57, and 60 ofregion c are identical in all eukaryotic tRNA genes; furthermore, there is only limited variation in positions GENERALIZED 8 lXLEOTIOES - RGYNNARYGG-31- 41 WCLEOTIDES --- NNTTCRNC--12ICLJoTofs 9, 15, and 56, which are always purines, and in positions 11 and SEQUENCE A -2IILOlE 16, which are always pyrimidines.t FIG. 6. Sequence comparison of the promoters of eukaryotic tRNA That these two regions and not other regularities in the se- genes. Sequences from tDNAW' and tDNALU are from this paper. quence of tRNA genes are recognized by the transcriptional tDNAMet is from Hofstetter et al. (5). The generalized sequence is de- machinery is also suggested by the fact that under the same rived from a comparison of all known eukaryotic tRNA sequences (8). conditions a prokaryotic tRNATYr gene fails to be transcribed When a nucleotide is indicated, it means that it is universally present. detectably in the Xenopus oocyte nucleus (15). Though this R, Y, and N stand for: always a purine, always a pyrimidine, and any bacterial tRNATYr gene shares other features with eukaryotic nucleoside, respectively. tRNA genes, it has a T-9 and a C-10 in its coding sequence in- stead of the purine-9 and G-10 universally found in eukaryotic be accomplished in a variety ofways, but in any case we would tRNA genes (8). expect that this flow ofinformation would be more difficult the longer is the distance to be covered. This fits well with the ob- We are thankful to Dr. Vincenzo Pirrotta for careful reading of the servation that the extent of transcription is inversely related to manuscript and for many useful criticisms and suggestions. G.C. was the length ofthe inserted DNA (Fig. 4), but it does not explain a Deutscher Akademischer Austauschdienst the absence of detectable transcript when regions a and c are ,supported by Fellowship. further apart than 140 nucleotides. If, on the other hand, the 1. Sakonju, S., Bogenhagen, D. F. & Brown, D. D. (1980) Cell 19, tRNA gene is not naked, but wound around nucleosomal beads, 13-25. then the physical distances between DNA regions may be 2. Bogenhagen, D. F., Sakonju, S. & Brown, D. D. (1980) Cell 19, shorter than when computed in a linear measurement along the 27-35. double helix. It is therefore possible that the RNA polymerase 3. Fowlkes, D. M. & Shenk, T. (1980) Cell 22, 405-413. III binds to two separated sites, by bridging the distance across 4. Guilfoyle, R. & Weinmann, R. (1981) Proc. Natl Acad. Sci. USA a and c in the 78, 3378-3382. the nucleosome. Because region region wild-type 5. Hofstetter, H., Kressmann, A. & Birnstiel, M. L. (1981) Cell 24, gene are separated by 40 nucleotides, they lie diametrically 573-585. opposite when wound around a nucleosome. As a consequence 6. Ciliberto, G., Castagnoli, L., Melton, D. A. & Cortese, R. (1982) both lengthening and shortening the sequence between them Proc. Natl. Acad. Sci. USA 79, 1195-1199. results in a decrease ofthe physical distance. Although the RNA 7. Rosenberg, M. & Court, D. (1979) Annu. Rev. Genet. 13, 319. polymerase III can still bind, the interaction would not be op- 8. Gauss, D. H. & Sprinzl, M. (1981) Nucleic Acids Res. 9, rl-r23. than 100 would 9. Weinmann, R. & Roeder, R. (1974) Proc. Nati Acad. Sci. USA 71, timal. However, inserts longer bp inevitably 1790-1794. bring either region a or region c outside the nucleosome, in 10. Koski, R. A., Clarkson, S. G., Kurjan, J., Hall, B. D. & Smith, which case polymerase binding becomes impossible and there M. (1980) Cell 22, 415-425. is no transcription. 11. Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) It is interesting that the interaction between tRNA genes and Nucleic Acids Res. 5, 4593-4611. nucleosomes has been shown not to be random. Wittig and 12. Cortese, R., Harland, R. & Melton, D. (1980) Proc. Nati Acad. Wittig (23) have provided evidence supporting aprecise phasing Sci. USA 77, 4147-4151. 13. Klein, R. D., Selsing, E. & Wells, R. D. (1980) Plasmid 3, 88-91. of nucleosomal particles in correspondence to tRNA genes. 14. Gronenborn, B. & Messing, J. (1978) Nature (London) 272, Analogous conclusions were reached for the 5S RNA genes (24, 375-377. 25). This nonrandom interaction between nucleosomes and 15. Melton, D. A. & Cortese, R. (1979) Cell 18, 1165-1172. tRNA genes may be an essential feature for transcription. 16. Laskey, R. A. & Mills, A. D. (1975) Eur. J. Biochem. 56, The results with the hybrid tRNALu-tRNAPrw gene show that 335-341. a and c are 17. Sutcliffe, J. G. (1978) Cold Spring Harbor Symp. Quant Biol 43, the essential regions independent transcriptional 77-90. signals regardless of the overall regularities of the structure of 18. Barrell, B. G. (1971) Prog. Nucleic Acids Res. 2, 751-779. tRNA genes. Apparently the only significant feature important 19. Rothstein, R. J., Lau, L. F., Bahl, C. P., Narang, S. A. & Wu, for their function is their sequence. A comparison of these se- R. (1980) Methods Enzymol. 68, 98-109. quences in homologous regions ofother eukaryotic tRNA genes 20. Chang, A. C. Y. & Cohen, S. M. (1978) J. Bacteriol 134, may be useful and is shown in Fig. 6. The three sequences 1141-1156. shown are those for which there is direct evidence for the func- 21. Saragosti, S., Moyne, G. & Yaniv, M. (1980) Cell 20, 65-73. 22. Sklar, V. E., Yamamoto, M. & Roeder, R. G. (1976) in RNA Poly- tion of region a and region c in transcription in the nucleus of merase, eds. Losik, R. & Chamberlin, M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 803-817. t The position number is that of the coding sequence of tDNAPr,; for 23. Wittig, B. & Wittig, S. (1979) Cell 18, 1173-1183. the other tRNA genes the position number of the equivalent nucleo- 24. Gottesfeld, J. M. & Bloomer, S. L. (1980) Cell 21, 751-760. tides may be different due to variations in the length of the D loop 25. Louis, C., Schedl, P., Semel, B. & Worcel, A. (1980) Cell 22, and extra arm of tRNA molecules. 387-392. Downloaded by guest on September 28, 2021