USAGE OF NONCANONICAL PROMOTER SEQUENCE BY THE YEAST MITOCHONDRIAL RNA-POLYMERASE

Prior work has demonstrated that a conserved nonanucleotide [5'-TATAAG TAA(+2)] promoter sequence is used by the mitochondrial [mt](1) RNA po lymerase in Saccharomyces cerevisiae. However, the highly AT-rich yeas t mt genome carries many other promoter-like sequences, but only a fra ction of them are involved in gene-specific transcription. To examine the sequence variability of this nonanucleotide promoter motif, single or multiple nt substitutions were introduced into the canonical promo ter sequence. The transcriptional activity of these altered promoter s equences was examined under the in-vitro reaction conditions. The resu lts presented here determined that several variant promoter sequences (i.e. TAAAAGTAA, TATAAGAAA, TATAAGTAG, TATAAGAAG, TATAAGAGA, TATAAGGGA , TATAAGTGG TAAAAGTAG) were efficiently used by the mtRNA polymerase. However, a single (i.e. AATAAGTAA, TTAAGTAA, TATTAGTAA, TATTACTAA, TAT AAGGAA, TATAAGTAT) or multiple (TATAGGAAA, TAAAAGGAA, TATAGGGAA, TAAAG GAAA, TAAAGGGAA) nt substitution(s) in other locations drastically red uced mt promoter function. Interestingly, some of these poorly or part ially active promoter variants (i.e. TATAAGGAA, TATAAGTAT, TATAAGTCA) became fully functional in the presence of sequence-specific dinucleot ide primer. Since dinucleotide primer bypasses the first phosphodieste r bond formation in transcription, it is suggested that the -1T-->G, 1A-->C and +2A-->T mutations affect mt transcription at the level of i nitiation rather than polymerase binding. (C) 1998 Elsevier Science B. V. All rights reserved.