IRESite record type: plasmid_with_promoter_and_putative_IRES_translationally_characterized
The shape of the nucleic acid molecule translated: linear
The quality of the mRNA/+RNA sequence: our_best_guess
The mRNA/+RNA description:
Putative in vivo bicistronic T7 transcript with CAT and GFP cistrons and 5'-UTR containing EMCV IRES
terminated by Tphi transcription terminator. The EMCV-R IRES is located between nucleotides 57-608 of the
putative mRNA and lacks the very rightmost 6 bases immediately preceding the initiator AUG codon of EMCV
polyprotein. A putative ERAV IRES is located in the intercistronic region and is retained only to the 4th
AUG codon (nucleotides 1964-1966) which starts the GFP ORF. Thus, there is no remaining sequence from
MCS between putative ERAV IRES and GFP ORF.
The mRNA/+RNA sequence represented in the +DNA notation:
Credibility of mRNA sequence: end-to-end_sequence_reverse_engineered_and_should_match_experiment
The description of the protein encoded in this ORF: green fluorescent protein artificially extended at its N-terminus by some 21 aminoacid residues initiated
from 2nd AUG codon encoded by ERAV IRES
The translational frameshift (ribosome slippage) involved: 0
The ribosome read-through involved: no
The alternative forms of this protein occur by the alternative initiation of translation: yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents): 1901-2767
It appeared the ERAV 5'-UTR region downstream the poly(C) tract contains 5 AUG codons. The first 4 are in
pairs adjacent to each other, while the fifth is the most downstream and is alone. Three types of proteins
were detected from complete ERAV IRES 1-961, called Lab-GFP, Lb-GFP, GFP. The naming Lab and Lb reflects
the fact that possibly viral proteases Lab and Lb are initiated at those respective AUG codons.
Plasmid constructs lacking a MCS between the cloned ERAV IRES and GFP ORF were described in Fig. 7A.
Results shown in Fig. 7C demonstrate that not all GFP protein fusion variants could be detected in
certain truncations of putative ERAV IRES. In this particular case, no plain GFP nor Lb-GFP was detected.
Authors claim the Lab-GFP corresponds in size to the one initiated in wild-type from AUG2, although chance
to distinguish from the polypeptide initiated at AUG1 which is longer by one aminoacid residue on 12%
PAA gel is questionable. It seems sequences downstream the second pair of AUG codons are required to allow
use of AUG codons 3 and 4.