Sequence architecture downstream of the initiator codon enhances gene expression and protein stability in plants

Nucleotide positions conserved on the 3' side of the initiator codon ATG and the corresponding N-terminal amino acid residues in a number of highly abundant plant proteins were identified by computational analysis of a dataset of highly expressed plant genes. The reporter genes uidA and gfp were modified to introduce these features. Insertion of GCT TCC TCC after the initiator codon ATG augmented expression for both the reporter genes. The insertion of each successive codon improved the expression of beta-glucuronidase (GUS) in an incremental fashion in transient transformation of tobacco (Nicotiana tabacum) leaves. The insertion of alanine-serine (Ser)-Ser resulted in about a 2-fold increase in the stability of GUS. However, this did not account for the 30- to 40-fold increase in GUS activity between the constructs coding for methionine-alanine-Ser-Ser-GUS and the native enzyme. Substitution of the codon for Ser at the third amino acid residue with synonymous codons reduced GUS expression. The results suggest a role for the conserved nucleotides in the +4 to +11 region in augmenting posttranscriptional events in the expression of genes in plants.