Abstract
The gene for ATPase subunit 9 of yeast mitochondria (Oli 1) contains two promoter sequences (Op1 and Op2) separated by 78 nucleotides. Both promoters are transcribed in vivo and in vitro though with different efficiency. The upstream promoter (Op1) is 12-15 times stronger than the downstream promoter (Op2), and this difference in promoter activity is partly attributable to the influence of the +2 nucleotide (Biswas, T. K., and Getz, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 270-274). In addition, the presence of the strong promoter (Op1) in close proximity to the weak promoter (Op2) partially inhibits the expression of the latter (Op2). The relative orientation of the two promoters has no influence on these inhibitory effects. When both promoters are present in the same reaction mixture, the strong promoter always competes effectively with the weak promoter for limited RNA polymerase (trans or competition effect). When the two promoters are present in the same plasmid, there is an inhibitory interaction between them that decreases as the distance between the two promoters increases (cis or position effect). Thus, the difference between the activities of a strong and a weak mitochondrial promoter in tandem is a function of two effects, the trans or competition effect and the cis or position-related effect. A model for promoter-promoter interactions is proposed.
Highlights
(Oli 1) contains two promoter sequences(Opl and Opz) and protein genes as well as of origins of replication
Both promoters are tran- We have recently shown that the+2 nucleotide influences scribed invivo and invitro though with different promoter function; the mitochondrial promoter with a purine efficiency
Theupstreampromoter (Opl) is12-15 nucleotide at position +2 acts as a strong promoter whereas times stronger than the downstream promoter (Opz), the same sequence with a pyrimidine nucleotide at +2 funcand this difference in promoter activity is partly at- tions as aweak promoter [7].The +2 nucleotide is the major
Summary
From the Departments of $Medicine, §Biochemistryand Molecular Biology, and lIPathlogy, University of Chicago, Chicago, Illinois 60637. The EcoRI- or HindIII-digested plasmid containing a synthetic promoter equivalent to either the yeast mitochondrial strong (Op,-like) or weak (Opt-like) promoter and the25-mer oligonucleotide containing either a strong or weak promoter sequence were mixed together and treatedwith DNA polymerase 1 (Klenow fragment) in thepresence of all four deoxynucleoside triphosphates. This allowed for the placement of the second promoter either upstream or downstream of the existing promoterand in eitheorientation of transcription. The band intensity of each transcript reflects the relative promoter strength
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