Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae.

摘要

In previous studies, we demonstrated the sufficiency of short nucleotide sequences to facilitate internal initiation of translation in mammalian cells. By using a selection methodology, we have now identified comparable sequences in Saccharomyces cerevisiae. For these studies, a library of constructs expressing dicistronic mRNAs with the HIS3 gene as the second cistron and 18 random nucleotides in the intercistronic region was introduced into a yeast strain in which the endogenous HIS3 gene was deleted. Untransformed cells or those containing the parent construct failed to grow on medium lacking histidine. Intercistronic sequences recovered from cells that did grow were evaluated by using various criteria. Fifty-six of the 18-nt sequences (approximately 1/400,000) functioned as synthetic internal ribosome entry sites (IRESes). The 14 most active sequences allowed growth in the presence of 0.1-0.6 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product. In addition, eight sequences were identified that were not IRESes, but that enhanced HIS3 expression by an alternative mechanism that depended on the 5' end of the mRNA and appeared to involve either shunting or reinitiation. Comparisons among the 56 selected IRESes identified eight significant sequence matches containing up to 10 nucleotides. Many of the selected sequences also contained extensive complementary matches to yeast 18S rRNA, some at overlapping sites. The identification of cis sequences that facilitate translation initiation in yeast enables detailed biochemical and genetic analyses of underlying mechanisms and may have practical applications for bioengineering.