Furthermore, although the Kozak consensus sequence has been incorporated in a multitude of expression studies, no in vivo validation has been done to correlate the Kozak sequence with functional expression levels. Nevertheless, since the initial characterization in 1987, little has been done to functionally characterize the natural sequences in vertebrates. This consensus sequence, now known as the Kozak consensus sequence, has become increasingly valuable as a tool for improving artificial expression constructs. However, characterization of nucleotide sequences surrounding known translational start sites revealed a DNA consensus sequence (5′-GCCRCC ATGG-3′) suggesting a conserved role in translational efficiency. In eukaryotes, targeting the small ribosomal subunit to the mRNA transcript is complicated through the addition of required protein: protein interactions. Originally characterized in 1974, the Shine-Dalgarno sequence is a consensus sequence (AGGAGGU) generally found immediately upstream of the translational start site. In prokaryotes, the small ribosomal subunit targets the initiation site through the base pairing of the 16s rRNA and the Shine-Dalgarno sequence on the mRNA transcript. For successful initiation, the small ribosomal subunit must successfully bind to the translational start site and recruit the large ribosomal subunit to form a functional ribosome. Both prokaryotic and eukaryotic translation progress through initiation, elongation and termination phases. The irreversible transfer of information from RNA to protein relies on ribosomal translation. The central dogma of molecular biology defines the transfer of information from DNA to protein through an RNA intermediate. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. Alfred Taubman Medical Research Institute Emerging Scholars program, funded by Mrs. Stern Career Development Professorship in Ophthalmology and Visual Sciences, and the A. AK would like to acknowledge the generous support of the Helmut F. All relevant data are within the paper and its Supporting Information files.įunding: This work was supported in part by R01 EY022633 from the National Institutes of Health, and utilized the Vision Research Core (P30 EY007003) at the University of Michigan. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: The authors confirm that all data underlying the findings are fully available without restriction. Received: JAccepted: AugPublished: September 23, 2014Ĭopyright: © 2014 Grzegorski et al. Furthermore, our results provide an experimental approach to testing and optimizing an important tool for molecular biology.Ĭitation: Grzegorski SJ, Chiari EF, Robbins A, Kish PE, Kahana A (2014) Natural Variability of Kozak Sequences Correlates with Function in a Zebrafish Model. We conclude that the canonical Kozak sequence is a poor predictor of translation efficiency in different model organisms. Rather, the most frequent natural variation of the Kozak sequence is almost twice as efficient. Our data reveal that while the canonical Kozak sequence is efficient, in zebrafish it is neither the most common nor the most efficient translation initiation sequence. Combining bioinformatics analysis with molecular biology techniques, and using zebrafish as a test case, we identify Kozak sequences based on their natural variance and characterize their function in vivo. Despite the critical importance of the Kozak sequence to regulation of gene expression, there have been no correlation studies between its natural variance and efficiency of translation. In eukaryotes, targeting the small ribosomal subunit to the mRNA transcript requires a Kozak sequence at the translation initiation site.