An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs

Kozak et al. (1987). Nucleic Acids Res DOI: 10.1093/nar/15.20.8125 Citations: 5189

A rough start-codon consensus had been proposed but untested across a large dataset. This paper compiled 5' sequences from 699 vertebrate mRNAs, defining start-codon context. 97% carry a purine at -3, and only 9% carry upstream ATGs, versus two-thirds of oncogenes.

Parts used

Kozak Sequence

Key findings

  • Analysis of 699 vertebrate messenger RNAs established (GCC)GCCACCATGG as the consensus sequence for translation initiation in vertebrates. Position -3 relative to the ATG start codon showed the strongest conservation with 97% containing a purine (61% adenine, 36% guanine, only 3% pyrimidine). Position +4 preferentially contained guanine. Site-directed mutagenesis experiments confirmed that mutations at positions -3 and +4 exert the strongest influence on translational efficiency.

  • Upstream ATG codons occurred in fewer than 10% of vertebrate mRNAs analyzed, with most containing only a single upstream ATG. A notable exception was proto-oncogene transcripts where nearly two-thirds contained upstream ATG codons, typically multiple, preceding the major open reading frame. Nonfunctional upstream ATG codons were frequently preceded by pyrimidines at position -3, contrasting with functional initiator codons that rarely had pyrimidines in that position.

  • Leader sequence length analysis of 346 vertebrate mRNAs with mapped transcriptional start sites revealed that most fell within 20-100 nucleotides. The distribution showed 29 mRNAs with 30-39 nt leaders, 36 with 40-49 nt, 38 with 50-59 nt, 37 with 60-69 nt, and 40 with 70-79 nt leaders. Only 4 mRNAs had leaders shorter than 10 nucleotides, while 68 had leaders between 100-199 nt and 19 between 200-299 nt, suggesting functional constraints on leader length.

1 3

Abstract

5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC) GCCA/GCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine, most often A, in that position. The periodical occurrence of G (in positions -3, -6, -9) is discussed. Upstream ATG codons occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the size range of 20 to 100 nucleotides. The significance of shorter and longer 5'-noncoding sequences is discussed.