Transcription initiation of eucaryotic transfer RNA genes.

Abstract

RNA polymerase Ill (Pal Ill), an enzyme found in the nuclei of animals, plants and fungi, has been implicated in the in vivo transcription of 5s rRNA, pretRNA, some small viral RNAs and the cellular RNAs derived from certain middle-repetitive genomic sequences. 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 experimental approach. In the first step, DNA is progressively 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 important for tRNA gene transcription initiation. Pol III Promoters Are lntragenic Deletion analyses have shown that the only DNA sequences essential for transcription of a frog 5s RNA gene are located between residues 50 and 83 (Sakonju 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 maximum 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 essential 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 approximate coordinates 8-19 and 52-62, respectively, by the standard system of numbering tRNA genes. Their locations with respect to the tRNA cloverleaf are indicated in Figure 1. Two lines of evidence in particular support the notion 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, 19211925, 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 sequences may still influence the efficiency of this process 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 transcription start point and the A block is less variable (10-I 6 bp). Another intriguing feature of these sequences is their close correlation with the most con-