The cyc1‐512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3′ untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1–512 mRNA contained many discrete 3′ termini at certain sites, ranging from the wild‐type position to over 2000 nucleotides (nt) downstream. The cyc1–512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base‐pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG…TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3′ endpoints, apparently identical to those seen in the cyc1–512 mRNA. However, several revertants contained minor end points not corresponding to any of the cyc1–512 mRNAs. The capacity of these three signals to form 3′ ends was confirmed with sequences constructed by site‐directed mutagenesis. We therefore suggest that the production of 3′ termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3′ mRNA termini, as represented by the cyc1–512 termini. The second type of element, which includes TAG…TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3′ preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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