POSITION-SPECIFIC INHIBITION OF YEAST MITOCHONDRIAL TRANSCRIPTION BY A POLY(T) SEQUENCE

The 3' flanking nucleotide(s) of the octanucleotide promoter sequence regulates transcriptional efficiency of some mitochondrial genes in Sa ccharomyces cerevisiae. To understand this regulation the in vitro tra nscription activity of various synthetic mitochondrial promoters carry ing different 3' flanking sequences was examined. The results presente d here demonstrate that consecutive thymidine residues, but no other p olynucleotides or secondary structure, in the promoter-proximal non-tr anscribed DNA strand inhibited mitochondrial transcription. The locati on and the number of T residues in the cluster as well as the concentr ation of UTP in the transcription reaction are the important factors d etermining this transcriptional inhibition. For example, a pair of thy midine nucleotides at positions +2 and +3 is sufficient for inactivati on of mitochondrial transcription, whereas more than three consecutive thymidine nucleotides beyond these positions are required for inhibit ion of mitochondrial transcription. However, a cluster of six to 12 th ymidine residues beyond position +11, a point where mtRNA polymerase h as been shown to form a stable transcription complex, did not interfer e with mitochondrial transcription. Interestingly, at low UTP concentr ation the mtRNA polymerase generates a large quantity of aborted initi ation products on a template carrying promoter-proximal poly(T) sequen ce probably due to the inability of the polymerase to clear this promo ter. On the other hand at high UTP concentration the same mtRNA polyme rase on the same mitochondrial promoter produces a higher level of pro ductive initiation complex. These observations suggest that the mechan ism of poly(T) inhibition of mitochondrial transcription is a UTP-limi ted transcriptional attenuation at the promoter site, which might occu r under specific physiological conditions (i.e. glucose repression-der epression, switching of aerobic-anaerobic conditions). (C) 1998 Academ ic Press Limited.