Nucleic Acids Research, Vol. 19, No. 21 5937-5942

In vitro transcription analysis of the region of Saccharomyces cerevisiae mitochondrial DNA containing the tRNAfM gene Tapan K.Biswas Wayne State University School of Medicine, Detroit, Ml 48201, USA Received July 24, 1991; Revised and Accepted October 3, 1991 ABSTRACT Prior work has indicated that an octanucleotide [5'TATAAGTA(+ 1)3'] sequence is used as a promoter in yeast mitochondria. Two such sequences (FPR and FP2) are present upstream of the tRNAfmt - RNase P RNA - tRNAPrW gene cluster but only the FP, promoter but not the FP2 appears to be active in vivo and in vitro. The results presented in this paper suggest that the downstream ATTAATT sequence close to the initiation site of FP2 causes premature termination of transcription and effectively inhibits transcription from the FP2 octanucleotide sequence. Thus the different levels of RNA synthesis from these tRNAfMet promoters might be determined by variable transcriptional initiation and elongation blockage events. Since FP, is found to be the only active promoter in this gene cluster, these three genes are thought to be transcribed together from the FP, promoter. In this study, a new promoter (SP) between the tRNAfmet and RNase P RNA genes has been identified which may participate in RNase P RNA gene expression. The sequence of the new promoter does not match perfectly to the mitochondrial conserved promoter sequence but does match to the consensus promoter sequence.

INTRODUCTION The 76,000 base pair circular genome of Saccharomyces cerevisiae mitochondria carries genes for two ribosomal RNAs (21S and 14S), 25 transfer RNAs, 8 proteins functioning in the respiratory chain, endoribonucleases involved in mRNA splicing and an RNase P RNA required for tRNA processing (1-3). Other mitochondrial proteins, including protein factors required for mitochondrial translation, transcription and replication, are coded by nuclear DNA and imported into mitochondria. Transcription of mitochondrial genome in yeast is well understood (4-17). Some mitochondrial genes are transcribed alone whereas other genes are transcribed as multi-genic transcripts which are subsequently processed into individual rRNAs, mRNAs or tRNAs. At least two protein factors, a 150 kDa core polymerase and a 43 or 70 kDa specificity factor are required for promoter recognition and gene specific transcription (6-8). Multiple sites of transcription initiation have been

identified on the yeast mitochondrial genome (4). A conserved eight nucleotide sequence [5'TATAAGTA(+ 1)3'] is present at each initiation site (4,5) and is found to be the only DNA sequence requirement for mitochondrial promoter function (9,10). 5'TAtAaGtN3' has been determined by site-directed mutagenesis

to be the minimum nucleotide combination that supports

mitochondrial promoter function (the lower case letters indicate positions at which nucleotide changes can be tolerated and N designates any nucleotide that can be used as an initiating nucleotide) (13). The presence of such promoter sequences at rRNA, tRNA and protein genes, as well as at the origins of replication, suggests that the same transcription machinery

transcribes all mitochondrial RNAs. It has been found that the nucleotide at position +2 controls the efficiency of transcription but does not affect the specificity of transcription initiation (11). A mitochondrial promoter with a purine at position +2 functions as a strong promoter whereas the same sequence with a pyrimidine at position +2 functions as a weak promoter (11). The low level of transcription from the weak promoter is due to the slow rate of formation of the first phosphodiester bond between a purine and a pyrimidine (14). The mitochondrial Olil and tRNAfmet genes each have two octanucleotide sequences in tandem (15,16). In both cases, the upstream sequence functions as a strong promoter whereas the downstream one is used poorly in the Olil gene (15) or not at all in the tRNAfmet gene under in vivo (4,17) or in vitro conditions (16). The upstream and downstream octanucleotide sequences are designated as FP1 and FP2 in the tRNAfmet gene, OP1 and OP2 in the Olil gene. Both the OP2 and FP2 sequences have a pyrimidine at position + 2 that in other promoters reduces efficiency of promoter function (11). A dinucleotide corresponding to positions + 1 and +2 enhances transcription from the weak promoter (OP2) of the Olil gene and brings it to the level of its strong promoter (0P1). In contrast, a dinucleotide corresponding to positions + 1 and +2 of the FP2 octanucleotide sequence does not activate its promoter function. Thus some other sequences, in addition to the pyrimidine nucleotide at position +2 must inhibit FP2 from functioning as a promoter. A DNA sequence comparison of the promoter regions of the Olil and tRNAfmet genes showed three differences in the sequences that flank the octanucleotide sequence: the space between the OP1 and OP2 (70 nucleotides) is larger than the space between the FPI and FP2 (3 nucleotides);

5938 Nucleic Acids Research, Vol. 19, No. 21 initiation site e.g., FP1-45 indicates that all mitochondrial nucleotides upstream of the 45th nucleotide from the initiation site of promoter FPI are deleted. For construction of the 3' deletion mutants, a 5' deletion mutant (FP1 +3) in which the FP1 promoter had been removed was used (Fig. 1B). The plasmid DNA was cleaved with Hinc II at the downstream sequence and then digested with exonuclease BAL-31 as described above. After digestion with Hind III and treatment with Klenow, the larger DNA fragment was isolated by 1 % agarose gel electrophoresis and the ends ligated with T4 DNA ligase. After transformation of E. coli, deletion mutants were isolated and the sequence of the 3' deletion mutants was confirmed by DNA sequencing. The 3' deletion mutant FP2+8 carries 19 nucleotides ATAATATAAGTATTAATTA mtDNA sequence including the FP2 octanucleotide (underlined nucleotides) (Fig. 2B).

FP2 but not OP2 is immediately upstream of several thymidine nucleotides and precedes a tRNA. To determine which of these differences account for the lack of FP2 acting as a promoter the region around FP2 was altered by deletion and substitution mutagenesis. The results presented in this paper demonstrate that the upstream strong promoter and the downstream tRNA do not have any role in inhibiting FP2 promoter function but the thymidine nucleotides close to the initiation site of FP2 do have an effect. In the course of these analyses, I have identified a new octanucleotide promoter (SP) between the tRNAfmet and RNase P RNA genes that could play a role in the regulation of the RNase P RNA gene.

MATERIALS AND METHODS Construction of deletion mutants Deletion mutagenesis was performed using a 393 bp mtDNA fragment in plasmid pUR 250 carrying a region of the tRNAfmet gene (62 bp upstream plus 331 bp downstream sequences from the initiating nucleotide of FPI promoter). For 5' deletions, the parent plasmid was cleaved at the 5' end with EcoR I (Fig. IA). This linear DNA was digested with BAL-31 for 15-90 sec at 300C and then treated with Klenow fragment to create blunt ends. The 3' end of the mtDNA was separated from the vector DNA by digestion with Hind HI. The mtDNA fragment was isolated by 6% polyacrylamide gel electrophoresis and then inserted into Hind IlIHinc II sites of plasmid pUR 250. The exact site of each deletion was determined by DNA sequencing (19) and are shown in Fig. 2A. The deletion mutants are designated with the total number of mitochondrial nucleotides present between the initiation site and the end of the deletion. A negative number indicates the position upstream from the initiation site and a positive number indicates the position downstream from the

Transcription reaction Plasmids containing the cloned mitochondrial promoters were linearized by digestion with appropriate restriction endonucleases and used as templates in the in vitro transcription reactions. The standard 25 ptl reaction mixture contained 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 20 mM KCl, 5% glycerol, 0.2 mg/ml rabbit serum albumin and 125 ;tM of each of the four rNTPs, 5 ACi of [&32P]-UTP and 40 Ag/ml DNA template. After addition of mtRNA polymerase, the reaction mixture was incubated at 30°C for 7 min and then the reaction was terminated by the addition of 25 yl stop solution (0.3% SDS, 200 jig/ml tRNA). Transcripts were separated from unincorporated label by precipitation with 50 kl of 5 M ammonium acetate and 250 yl of ethanol. These RNAs were resolved on 5 % polyacrylamideurea (8 M) gels and then visualized by autoradiography. The RNA products were quantitated by scanning the autoradiograms

A E

H

Hc

E

Hc

VI

H

sp

IPP

H

p

and 1. EcoRl digestion 2. BAL-31 digestion 3. Klenow treanent 4.

HisdIII digestion and religation of the smaller DNA fragment into HinclttHindlll sites of a plasmnid

S. Isolation

pUR 250 and then transformed into E. coli

B P

Hc

H

> 2

1

SP

P

P

-),

P

w

1. HincIl digestion 2. BAL-31 digestion 3. Hisdll digestion 4. Klenow treament

S. Isolation and religation of the larger DNA fragmnent and then transformed into E. cols

Fig. 1. Representation of the deletion mutagenesis of the tRNAfmel gene promoters. A pUR 250 plasmid bearing a 393 bp region of the mitochondrial tRNAfme gene was used for deletion study. A: construction of 5' deletion mutants; B: construction of 3' deletion mutants. The procedure for deletion mutagenesis is described in 'Materials and Methods' section. Restriction site abbreviations used: E, EcoR I; H, Hind III; Hc, Hinc II; P, Pvu II.

Nucleic Acids Research, Vol. 19, No. 21 5939 using a densitometric scanner (Hoefer Scientific Instrument). Different exposures were used to establish a linear range of signal intensity.

RESULTS Transcription from the octanucleotide sequences upstream of the tRNAftet tandem promoters was studied in vitro. Fig. 2A shows the locations of the octanucleotide sequences in the tRNAf¶et gene which are separated by 3 nucleotides. If both sequences can act as promoters, then two different transcripts are expected from the tRNAfIet template. Products from the in vitro transcription of the Hind III digested tRNAfIet template are shown in Fig. 3A. A 337 nucleotide transcript from the FP1 promoter was detected whereas a 326 nucleotide RNA that would have been initiated from the FP2 sequence was not found (Fig. 3A, lane 2). An additional transcript of 143 nucleotides that originated from another downstream promoter was identified (see below). The activity of the FP2 sequence was further examined by a similar experiment in which shorter transcripts should be produced (Fig. 3B, lane 2). A 49 nucleotide and a 38 nucleotide transcript were expected if both the FP1 and FP2 sequences were active as promoters on a Hinc II digested template. The 49 nucleotide transcript but not the 38 nucleotide transcript was found suggesting that the FP2 octanucleotide sequence is not active. Since a dinucleotide corresponding to positions + 1 and +2 is used as a primer by mtRNA polymerase (20) and bypasses the step of first phosphodiester bond formation (14), the effect of the dinucleotide on transcriptional activities of the tandem A AFP1-58 S'TTTTTTATTT ATTATTTTTA ATTAGTAAAA ATTATATTAT ATATATATAT ATTAAATTTT

octanucleotide sequences of the tRNAtmet gene was examined (Fig. 4, lanes 1-4) . For comparison, the tandem promoters (OP1 and OP2) of the mitochondrial Olil gene were also used (Fig. 4, lanes 5-9). The OP1 and OP2 promoters are separated by 70 nucleotides. At a suboptimal concentration (

In vitro transcription analysis of the region of Saccharomyces cerevisiae mitochondrial DNA containing the tRNA(fMet) gene.

Prior work has indicated that an octanucleotide [5'TATAAGTA(+1)3'] sequence is used as a promoter in yeast mitochondria. Two such sequences (FP1 and F...
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