Utilizing Ribosome Frameshifting Mechanism to Express Downstream Genes Using a Single Ribosome Binding Site

Abstract

Translation initiation is a rate-limiting and highly regulated step in protein synthesis in both prokaryotes and eukaryotes. It comprises the ribosomal recognition of start codon of mRNA and ensuring the alignment of ribosome with the correct reading frame. Ribosome binding site, a specific region on mRNA strand facilitated in correct positioning of ribosome with the start codon of mRNA for efficient and accurate translation thereby regulating the gene expression and production of correct proteins. The study is designed by considering the potential translation initiation of both upstream and downstream genes using single ribosome binding site. A genetic construct utilizing a single ribosome binding site was used to initiate the translation of both upstream and downstream genes and through this approach aimed to determine the protein expression of downstream gene. Stop/start overlap patterns and varying spacer lengths in between upstream and downstream genes were used in the genetic constructs. The protein expression levels were determined through sodium dodecyl sulfate- polyacrylamide gel electrophoresis and enzyme activity assays. The findings of the study imply the expression of downstream gene suggesting the involvement of ribosomal frameshifting enable the accessibility of start codon for translation initiation of downstream gene.