Cell, Vol. 29, 3-5. May 1982, Copyright 0 1982 by MIT

Transcription Initiation of Eucaryotic Transfer RNA Genes

Benjamin D. Hall Department of Genetics University of Washington Seattle, Washington 98195 Stuart G. Clarkson Department of Microbiology University of Geneva Medical School 1205 Geneva, Switzerland Glauco Tocchini-Valentini Laboratory of Cell Biology National Council for Research 00196 Rome, Italy

RNA polymerase Ill (Pal Ill), an enzyme found in the nuclei of animals, plants and fungi, has been impli- cated in the in vivo transcription of 5s rRNA, pre- tRNA, some small viral RNAs and the cellular RNAs derived from certain middle-repetitive genomic se- quences. It is a complex -700 kd protein composed of at least ten distinct subunits. The enzyme is not able to transcribe purified genes with fidelity by itself but requires additional components whose number, nature and modes of action are only just beginning to be characterized. Much more is known about the locations of the DNA signals that permit accurate transcription initiation of some Pol Ill genes. This information has come primarily from a two-step ex- perimental approach. In the first step, DNA is progres- sively deleted from around and within the gene in question; the deleted sequences are then replaced by heterologous DNA. Alternatively, single-base changes or short deletions are introduced into the gene by in vivo or in vitro mutagenesis. The second step is then to assay the transcriptional effects of these sequence manipulations by microinjection of the mutant genes into the nuclei of frog oocytes or by their incubation in a variety of in vitro transcription systems. We shall compare and contrast the results of such analyses with several Pol Ill genes and discuss, in particular, the structure and function of the DNA signals impor- tant for tRNA gene transcription initiation. Pol III Promoters Are lntragenic Deletion analyses have shown that the only DNA se- quences essential for transcription of a frog 5s RNA gene are located between residues 50 and 83 (Sak- onju et al., Cell 79, 13-25, 1980; Bogenhagen et al., Cell 79, 27-35, 1980). A plausible basis for the mode of action of this intragenic promoter was provided by Engelke et al. (Cell 79, 717-728, 19801, who showed that the same region of the 5s RNA gene binds to a 5S-specific transcription factor. These results suggest that this 37 kd transcription factor, once bound, may interact with a Pol III molecule to position its catalytic sites on the transcription start point of the DNA.

The same resection approach has shown that tRNA genes also contain intragenic promoters. Their maxi- mum boundaries have been defined as residues 8 and

62 within the genes encoding the initiator tRNAMe’ (Hofstetter et al., Cell 24, 573-585, 1981) and tRNA% (Galli et al., Nature 294, 626-631, 1981) of X. laevis and the tRNAP”’ of Caenorhabditis elegans (Ciliberto et al., PNAS 79, 1195-l 199, 1982). Unlike their counterparts within 5s genes, however, the es- sential nucleotides are split into two sequence blocks that are set far apart. These sequences, termed the A and B blocks (Galli et al., op. cit.), have the approxi- mate coordinates 8-19 and 52-62, respectively, by the standard system of numbering tRNA genes. Their locations with respect to the tRNA cloverleaf are in- dicated in Figure 1.

Two lines of evidence in particular support the no- tion of discontinuous intragenic promoters: chimeric tRNA genes containing the 5’ half of one gene and the 3’ half of another can be transcribed well; and transcription can also occur after the replacement of the central region of tRNA genes with DNA of very different sequence (Ciliberto et al., op. cit.; Galli et al., op. cit.). These central regions do appear to have a spacing function, however, because the efficiency of transcription depends on the length of the replaced DNA, the optimal distance between the A and B blocks being 30-40 bp (Ciliberto et al., PNAS 79, 1921- 1925, 1982). In natural tRNA genes this distance can vary from 31 to >74 bp, the variability being due to the length of the V arm and the presence within certain tRNA genes of an intervening sequence. Although not absolutely required for transcription (Wallace et al., Science 209, 1396-1400, 1980), intervening se- quences may still influence the efficiency of this proc- ess by expanding the distance between the A and B blocks, thereby diminishing promoter strength.

An unusual feature of tRNA gene promoters is thus the great latitude in distance between the A and B blocks. In contrast, the distance between the tran- scription start point and the A block is less variable (10-I 6 bp). Another intriguing feature of these se- quences is their close correlation with the most con-

1 3’

0 arIm r ------- ia

- - - - -

Cell 4

served regions of bacterial and eucaryotic tRNAs: the invariant nucleotides U8, A14, G18 and Gl9 are encoded by the A block, while G53, T55, C56, A58 and C61 are found in the B block. Point mutations at C56 of a yeast tRNATy’ gene reduce or abolish its transcription in vitro (Koski et al., Cell 22, 415-425, 1980). Hence some invariant nucleotides may have been conserved within eucaryotes because of their importance for transcription initiation, as well as their importance for tRNA functions such as interaction with aminoacyl-tRNA synthetase and ribosome bind- ing. Indeed, it is even conceivable that some of these conserved nucleotides are not actually needed for tRNA functions.

Pol Ill thus has to cope with at least two distinct kinds of intragenic promoters, one for 5s RNA genes and another for tRNA genes. The second category appears to include also the mammalian Alu family of middle-repetitive sequences and the genes encoding the adenovirus 2 VA RNAs and the Epstein-Barr virus EBER RNAs. Sequences closely homologous to the conserved regions of tRNA genes are included within the region of the adenovirus 2 VAI RNA gene that is essential for transcription (Fowlkes and Shenk, Cell 22, 405-413, 1980; Guilfoyle and Weinmann, PNAS 78, 3378-3382, 1981). In addition, tRNA genes are efficient competitors of VA RNA transcription, whereas 5s RNA genes are not. Finally, transcription of the VAI and tRNA genes each requires the same protein fractions, but neither requires the 37 kd poly- peptide specific for 55 RNA transcription. Promoter function -Linear Sequence or Conformation? If the A and B blocks within tRNA genes can indeed be viewed as the prototype of a second category of Pol Ill promoter, then how do they function? It is generally assumed that the A and B blocks interact with one or more transcription factors, rather than with the polymerase directly. This seems reasonable in view of the inability of Pol Ill to transcribe purified DNA templates accurately. In addition, while the 5’ and 3’ halves of an X. laevis tRNAyet gene and of the adenovirus 2 VAI RNA gene are unable to direct transcription in vitro, only the 3’ half of each gene can inhibit transcription of the intact gene (Kressmann et al., NAR 7, 1749-1763, 1979; Fowlkes and Shenk, op. cit.; Guilfoyle and Weinmann, op. cit.). This ability is lost in deletion mutants that lack the conserved nucleotides of the B block. On the basis of these results, we propose that the primary interaction re- quired for transcription of both tRNA and VAI RNA genes is the binding of a transcription factor to their B block sequences. The A blocks of tRNA genes, and perhaps also the VAI gene counterpart, are then rel- egated to a positioning role; that is, their function is to position Pol Ill at a purine 1 O-l 6 bp farther upstream, where it then initiates transcription.

What kind of recognition signals permit the binding of a transcription factor to tRNA genes? No mecha-

nistic proposal has yet been advanced to explain the interaction between a transcription factor and any particular B block sequence. The interaction may simply involve, as for repressor binding to the Esche- richia coli lac operator, direct protein binding to dou- ble-helical DNA. In this case, the 5 invariant nucleo- tides within the I3 block (G53, T55, C56, A58 and C61) are likely to specify the essential contact points for factor binding.

We wish to propose an alternative model, which ascribes different functional roles to discrete subseg- ments of the B block. It embodies the fact that the essential A and B blocks are precisely those regions that encode single-stranded loops (the D and T loops) that, in every tRNA molecule, engage in a strong mutual tertiary interaction. Specifically, as shown by the dashed lines in Figure 1, tertiary base pairs are found between G18 and #55, and between G19 and C56. It is also striking that base-paired regions of the T stem are encoded by the B block, whereas the A block sequences encode only the 5’ portion of the D stem, together with the invariant nucleotides U8 and A14, which form an additional tertiary base pair in tRNA. We propose that the B block DNA sequences exist in an intrastrand stem-and-loop conformation when a transcription factor binds to a tRNA gene, and that this binding promotes tertiary interactions be- tween sequences encoding some of the conserved nucleotides of the D and T loops. The overall result is that the plus strand of the DNA has a three-dimen- sional conformation that at least partly resembles that of the RNA transcript. According to this hypothesis, relatively few nucleotides of the A and B blocks are required per se as sequences that transcription fac- tors recognize. Instead, some of the B block se- quences are needed as regions of dyad symmetry, while other conserved nucleotides of both blocks are required for mutual tertiary interactions.

This model may help to explain some apparently paradoxical results of point-mutation analyses. Sub- stitution of a G or T for the invariant C at position 56, which precludes or weakens the proposed Gl g-C56 tertiary interaction, severely reduces in vitro transcrip- tion of a yeast tRNATY’ gene (Koski et al., op. cit.). In contrast, a G57 to T57 substitution has no adverse effect on transcription of a human tRNAyef gene, al- though processing of the tRNA precursor is blocked (Zasloff et al., Nature 295, 533-535, 1982). This is also compatible with the model, because no tertiary pairing is envisaged for this nucleotide. More posi- tively, we predict that transcription will be severely reduced by the transversion T55 to G55, whereas a C at this position may even enhance it. By the same reasoning, we expect transcription to be inhibited by a pyrimidine substitution at either G18 or G19, but not by a G18 to Al8 transition. Two point mutations already described within the A block indeed have strikingly different effects: a T8 to G8 transversion within a Drosophila tRNAFg gene abolishes transcrip-

Cell 5

tion (Sharp et al., PNAS 78, 6657-6661, 1981) whereas it is unaffected by an Al 4 to Gi 4 transition within a yeast tRNATy’ gene (Koski et al., op. cit.). This may indicate that a T8-A14 interaction in the DNA plus strand is also important for transcription initiation.

The tertiary interaction model makes several other predictions whose testing will require more complex sequence manipulations. For example, a chimeric tRNA gene that is unable to form a T stem should be very poorly transcribed even if it contains all of the conserved nucleotides of the B block. A construction of this type has not yet been reported, but 4 bp have been deleted from the 5’ portion of the T stem of a C. elegans tRNAP” gene (Ciliberto et al., PNAS 79, 1 195-1199, 1982). This deletion, which eliminates the T stem except for the invariant G53-C61 base pair, reduces transcription by a factor of 100.

A more basic premise is that transcription will be abolished by the elimination of the entire B block. Two reports have recently appeared, however, that at face value do not support this assumption. Sharp et al. (op. cit.) created deletions from the 3’ end of a Drosophila tRNApg gene to define the 3’ boundary of its intragenic promoter. They obtained the surprising result that transcription was merely reduced after removal of the consensus B block sequences, and that it was only abolished when deletions reached the 3’ end of the D stem. Carrara et al. (Cell 27, 371-379, 1981) inserted 300 bp of mouse DNA into the intervening sequence of a yeast tRNA? gene. This expanded gene was efficiently transcribed in vitro, even after removal of part of the mouse insert together with the 3’ half of the gene. interpreted most simply, these results sug- gest that just the A blocks are needed for the expres- sion of these two tRNA genes. This interpretation, however, runs counter to the evidence that demon- strates a requirement for the B block sequences in the transcription of other eucaryotic tRNA genes.

One way out of this dilemma is to regard all of these 3’-deleted tRNA genes as substitution rather than deletion mutants. This leads to the alternative inter- pretation that the sequences used to replace the 3’ halves of the Drosophila tRNAFg and yeast tRNA2” genes are able to substitute functionally for the B blocks, whereas the replacement sequences used in other 3’-deleted tRNA genes lack this ability. The normal D and T arms of the Drosophila tRNA$‘g gene, for example, are shown in Figure 2 in a stem-and-loop conformation, with circles around the invariant nucleo- tides G18, G19, T55 and C56. In the 3’ deletions made by Sharp et al. (op. cit.) this T arm was replaced by pBR322 DNA. A striking feature of this ptasmid sequence is its potential to form a substitute T arm, complete with T and C residues in the loop (Figure 3). Stem-and-loop structures can be drawn similarly for the mouse DNA used in the insertion-deletion exper- iments of Carrara et al. (op. cit.). We propose that transcription can proceed in both cases because of

the availability of such substitute 6 blocks, which are able to form the necessary structure for factor binding and for tertiary interactions with the normal A block sequences. Transcription Modulation We have so far considered only the intragenic se- quences that are essential for transcription initiation of eucaryotic tRNA genes. But the transcriptional ac- tivity and starting specificity of a given tRNA gene depend also on the nature of the upstream DNA se- quences. The normal V-flanking sequences of a silk- worm tRNAA” gene, for example, are absolutely re- quired for expression of this gene in a homologous cell-free extract (Sprague et al., Cell 22, 171-l 78, 1980). Deletions and substitutions in the Y-flanking sequences of other tRNA genes can reduce their transcription appreciably, and may also change the frequency of use of a particular initiating nucleotide. Variations in 5’-flanking sequence that affect tran- scriptional activity also occur naturally within tRNA gene families, implying that these upstream se- quences may act as a fine-tuning control. In some cases, this control is exerted in a negative way. For example, a G+T-rich undecanucleotide that precedes several Drosophila tRNALyS genes is inhibitory for tran- scription when located 13 nucleotides in front of the mature tRNA-coding sequence (DeFranco et al., JBC 256, 12424-l 2429, 1981). A different kind of inhib- itory sequence is found 11 bp in front of a variant tRNA?’ gene of X. laevis (Clarkson et al., ICN-UCLA Symposia 23, 463-472, 1981). The special feature of this sequence, a 9 bp G+C-rich alternating purine- pyrimidine tract, is its potential for forming lefthanded DNA.

Transcription initiation of eucaryotic tRNA genes thus appears to depend critically on DNA conforma- tion, both within and around the genes. What is now required, clearly, is progress in the isolation of the additional components needed for accurate gene expression. Their availability will permit a test of some of the predictions of the tertiary interaction model and, more important, a direct analysis of the DNA confor- mation of actively transcribed tRNA genes.

2 ,@@A A T

3 ;m,

G T O*

0 T n T

0 A ptm322 A A D arm c c ‘T~W.l

c A C-G G--c G-C C -G G-C

G ,, T 5’-T G

G-C G ---m-m bp ---A A Ts