There are 638 nucleotides between the initiation codons (ATG) of these two genes.
In the transcripts of C/EBPα and β, a uORF is located out of frame between the initiation codons of the extended (α-ext and LAP*) and the full-length isoforms (p42 and LAP) 43 (Fig. 2).
However, the distribution of motif 2 is relatively flat, with ~70% of the sequences located upstream of the TSS and the remaining 30% located between the TSS and the initiation codon.
Although the mechanism of the inhibitory effect on translational activity by spacing remains to be elucidated, it appears that nucleotide spacing between the enhancer and the initiation codon can sometimes affect translational activity.
While replacing mCherry with TdTomato, we destroyed a Kozak consensus sequence between the myristoylation sequence and the initiation codon of mCherry, in order to increase the probability that translation would initiate at another Kozak site upstream of the two myristoylation sequences, thereby reducing possible cytoplasmic 'background' localisation.
The extra base pair to disrupt reporter expression arising from translation from further upstream, had to be inserted immediately between the splice acceptor and the initiation codon and, therefore, could have perturbed expression for transcripts b/c more dramatically, as for fkh-9b.
Interestingly enough, the pattern exhibited considerably agreement in spite of the fact that the DNA sequences in the 5' UTR located between the sites of splicing and the initiation codon were poorly conserved.
Because the distance between the 5′-translational enhancer and the initiation codon is shorter in the Flexi ORF clone than in the Gateway ORF clone, these results suggest that the Flexi ORF clone could be the clone of choice to consistently obtain high protein productivity in both the TD and WGEP systems.
Along the same line, we also compared orthologous genes between T. brucei and T. vivax to investigate whether the spatial pattern of trans-splicing sites, namely their number and distances to the initiation codon, was similar between these two African parasites.
An 8-nt overlap occurred between the termination codon of ORF1b and the initiation codon of ORF2.
