p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53 alpha isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53 alpha isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53 alpha isoforms.