No abstract

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [a-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above-i.e., 20-40 nucleotides upstream of the tRNAPhe gene-had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.Previous studies on mtDNA transcription in HeLa cells have shown that both strands are completely or almost completely transcribed (1,2). This conclusion has been recently confirmed for the heavy (H) strand by the observation that the discrete transcripts of this strand form a continuum extending over its entire length, with the exception of a relatively small region from coordinate 98/100 to coordinate 5/100 [relative to the origin ofreplication taken as 0/100 (3-5) (Fig. 1)]. In the present work, the initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mtDNA have been investigated by an approach that has been used successfully for identifying the transcriptional initiation sites in yeast mtDNA (8,9). In particular, the 5' ends oftotal poly(A)-containing mitochondrial RNA possessing a di-or triphosphate, and therefore presumably resulting from transcriptional initiation, have been identified by taking advantage of the capacity of the guanylyltransferase ("capping" enzyme) to transfer GMP from GTP to di-or triphosphate-terminated polynucleotides (10- (6) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

We have examined the initiation of transcription of the mitochondrial genes for ribosomal RNA (rRNA) in the yeast Kluyveromyces lactis and show that these are transcribed independently from individual promoters. The mature large rRNA contains a 5' di- or triphosphate end which can be labelled in vitro with [alpha-32P]GTP using guanylyltransferase and this enabled us to determine the nucleotide sequence of its 5' terminus. For the small rRNA, a minor in vitro capped RNA species hybridizes in the region where--as judged from S1 nuclease protection experiments--the precursor of this RNA starts. We have determined the DNA sequence around the beginning of both rRNA genes and this reveals the existence of an identical nonanucleotide sequence (5' -ATATAAGTA- 3') just preceding the positions where the rRNAs start. This sequence is identical to the one preceding the rRNA genes in the mtDNA of the distantly related yeast Saccharomyces cerevisiae (Osinga, K.A. and Tabak, H.F. (1982) Nucl.Acids Res. 10, 3617-3626) and supports our proposal that this sequence motif is part of a yeast mitochondrial promoter. We have noticed that the same sequence is located in the putative origin of replication present in hypersuppressive petite mutants of S. cerevisiae and consider the possibility that this sequence is involved in RNA priming of DNA replication.

Mammalian mitochondrial genomes have a presumptive transcription termination site at the 16S rRNAtRNALU gene boundary. We have developed an in vitro system from human KB cells that terminates transcription at this gene boundary. By S1 nuclease protection, the 3' ends of terminated transcripts were mapped 3 and 4 base pairs upstream of the 16S rRNA-tRNAL" gene boundary, in agreement with in vivo data.By high-resolution sizing of transcripts, the 3' end was mapped 7 ± 1 base pairs downstream from the gene boundary. Termination occurs with equal efficacy from transcriptional initiation at the heavy-or light-strand promoter. AU template nucleotide sequence information necessary for termination appears to be located near the termination site itself. An unexpected observation is that the termination region functions bidirectionally.Transcription of mammalian mtDNA initiates from promoters in the displacement (D)-loop region, producing polycistronic RNA precursors that are processed into mature transcripts by cleavages between the gene sequences (1). Two lines of evidence indicate that transcription of the heavy (H) strand can either terminate at the 3' end ofthe 16S rRNA gene or continue transcribing H-strand genes. First, the ribosomal gene region appears to be transcribed 50-100 times more frequently than the other H-strand genes (2). Second, the heterogeneity of the 3' ends of 16S rRNA of several species (3-5) suggests that 16S rRNA termini may be the result of transcription termination, rather than a precise processing likely responsible for the 3' ends of most mature mitochondrial transcripts. We describe here an in vitro system derived from human mitochondria and show promoter-independent, bidirectional termination at the 16S rRNA-tRNAL"u gene boundary. MATERIALS AND METHODSClone Construction. A Hae II fragment inserted into pACYC177 (6) supplied mtDNA from nucleotides (nt) 3160 to 4534 (7). Clone L3'A-91, containing the mtDNA sequence from nt 495 to 741, and L5'lA-28 with the mtDNA sequence from nt 1 to 440, provided the promoter sequences (8). From this source material, three template clones were constructed with standard techniques (9). Clone A consists of nt 495-739, a 16-base-pair (bp) linker, and nt 3165-4121, all inserted between the BamHI and EcoRI sites of pBR322. Clone B is similar, except that nt 440-324 and a 10-bp linker precede the nt 3165-4121 fragment. Clone C consists of nt 440-324joined without linkers to nt 3316-3165, all inserted between the BamHI and EcoRI sites of pBR322.Transcription Termination Reactions. For the transcription termination reactions, mtRNA polymerase was prepared as described (10). The transcription mixture was typically in a 50-1.l vol containing 10 mM Tris HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, bovine serum albumin at 100 pg/ml, 180 ,M CTP and UTP, 360 AuM ATP, 0.01 AM [a-32P]GTP (3000 Ci/mmol; 1 Ci = 37 GBq), 0.5 ,ug of DNA (10 gg/ml), and 7-14 A1 of the polymerase preparation; reactions were incubated at 15°C for 45 min and terminated by adding 10,ul o...