Signals determining translational start-site recognition in eukaryotes and their role in prediction of genetic reading frames

Abstract

A special methionyl-tRNA (RNA_i) is universally required to initiate translation. The conservation of this reactant throughout evolution, as well as its unusual decoding properties, suggested an alternate mechanism for tRNA-mRNA interactions at initiation. We have reported that the sequence of bases neighboring the start codons of many eubacterial genes are complementary not only to the 16S rRNA 3′ end and to the anticodon of tRNA_i, but, also, have the potential to base-pair the D, T or extended anticodon loops of this tRNA_i. The coding properties of tRNA_i and mutations that affect translation suggest that these signals may function. This hypothesis explains the observation that unusual triplets can start prokaryotic and mitochondrial genes and predicts the occurrence of other reading frames. Furthermore, it suggests a unifying model of chain initiation based on RNA-RNA contacts and displacements.

Here we examine the start domain of 290 eukaryotic genes for their ability to base-pair the tRNA_i loops and the 18S rRNA. We observe that both methionine start, and methionine coding regions have the potential to pair with the 18S rRNA, but that the nucleotide distribution about start codons strongly favoured such pairings over that near internal AUGs. The 5′ extended anticodon of tRNA_i is methylated, and was not represented in the mRNA with high frequency. However, the tetramer AUGg did occur with high frequency in the start domain. A modification of the tRNA_i T loop also decreases its base-pairing potential. Interestingly, complementarity to the T loop did not occur with high frequency in the start sites. The early coding region, 10 to 34 nucleotides 3′ to the initiator AUG, is complementary to the tRNA_i D loop in many cases, while no such affinity is found near internal AUGs.

The nucleotides around initiator AUGs were heavily biassed toward the sequence gccaccAUGgcg. No such tendency was noted around internal AUGs. Although the role of this sequence bias is unclear, the sequence gccaccAUGg has been shown by Kozak to promote initiation. Another distinguishing feature was a C-rich tract 7 to 34 nucleotides 5′ to the initiator AUGs.

Ability to pair with more than eight bases of the start consensus sequence, matching of 6 or 7 nucleotides to the D loop on the 3′ side, an C-richness on the 5′ side were used as criteria for distinguishing start AUGs. The program successfully identified over 52% of the sequences submitted to it, wrongly identified less than 4% and labelled the rest as uncertain suggesting a promising approach to reliable detection of eukaryote genetic reading frames.