A Complex Unidirectional Signal Element Mediates GCN4 mRNA 3′ End Formation in Saccharomyces cerevisiae

Abstract

The yeast GCN4 3′ element represents a class of polyadenylation sites which function unidirectionally and efficiently in test systems in vivo as well as in vitro. A complex signal element is required for polyadenylation activity with a minimal size of 116 nucleotides for the functional element. We subdivided this element into five regions (EL1 to EL5) of 16 to 26 nucleotides each. Each region was characterized by deletion analysis in an in vivo test system. Two TTTTTAT motifs are located in different regions (EL1 and EL4) upstream of the poly(A) site. The 3′ end processing activity was significantly reduced when both motifs were mutated by site-directed mutagenesis and abolished when EL1 and EL4 were deleted. The major poly(A) site is located in EL5, 3 nucleotides downstream of the second TTTTTAT motif. Additional minor poly(A) sites are used in less than 10% of the mRNA 3′ ends. Deletion of EL3 resulted in a changed pattern of mRNA 3′ ends by increased usage of the minor poly(A) addition sites. The major poly(A) site in EL5 can be removed without loss of function when sequences upstream of EL1 are present. The tripartite TAG…TATGT…TTT sequence located downstream of EL5 is not required for function.