PURINES ARE REQUIRED AT THE 5'-ENDS OF NEWLY INITIATED RNAS FOR OPTIMAL RNA-POLYMERASE-III GENE-EXPRESSION

We have made specific alterations in the CAACAA element at the transcr iption start site of a Saccharomyces cerevisiae suppressor tRNA gene, The mutant genes were tested for their ability to suppress the ochre n onsense alleles ade2-1, lys4-1, and met4-1, Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding th e +1 and +2 nucleotides of pre-tRNA(Tyr), had a strong deleterious eff ect in vivo, as did the more extensive change CTCCTC, In contrast, mut ant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels, The transcription start point cou ld be shifted in a limited fashion in response to the specific sequenc es encountered by RNA polymerase III at the start site, ATP was prefer entially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide, Short oligopyrimid ine RNAs formed on the CTCCTC allele could be repositioned in the acti ve center of the newly formed ternary complex, Early postinitiation co mplexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive t ranscripts than similar complexes formed on SUP4-o, Our results sugges t that the purine-rich sequences at the 5' ends of the nascent transcr ipts of many genes act to stabilize the early ternary complex.