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 with its 1st, 2nd and 4th AUG
mutated from AUG to AUA.
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 extended at its N-terminus by 19 aminoacid residues encoded by 3'-end of ERAV IRES
and partly by multiple cloning sequence site (as if initiated from AUG3).
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): 1961-2821
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 245-961, 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.
In this particular construct the AUG1, AUG2 and AUG4 were mutated to AUA (Fig. 5B).
The IRES name: ERAV_245-961_deltaA1_deltaA2_deltaA4
The functional status of IRES: functional
The IRES absolute position (the range includes START and STOP codons or their equivalents): 1290-2004
How IRES boundaries were determined: experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF: 16
3'-end of IRES relative to last base of the STOP codon of the upstream ORF: 730
5'-end of IRES relative to first base of the START codon of the downstream ORF: -671
3'-end of IRES relative to first base of the START codon of the downstream ORF: 43
The sequence of IRES region aligned to its secondary structure (if available):
This mutation confirms that Lab-GFP was synthesized from AUG1 and AUG2 whereas AUG3 and AUG4 are be used to
initiate Lb-GFP and that AUG3 can be to some extent used to initiate Lb-GFP, albeit inefficiently.
The relative translation efficiency in % of this IRES: 82.000
Name of IRES used as the positive control: ERAV_245-961
Name of the plasmid used as the positive control. pE1(245-961)
IRESite Id of the plasmid used as positive control. 272
The relative translation efficiency in % of the positive control: 100.000
The size (length) of intercistronic region in the positive control: 626
The effect of 5'-cap analogs on translation: not tested
Rapamycin affects translation: not tested
Type of RNA subject to translation: endogenous_cytoplasmic_uncapped_T7_transcript_without_polyA_tail
Mutation of 1st, 2nd and 4th AUG codon contained within the putative ERAV IRES only decently impaired
IRES activity. The various GFP-fusion protein yields changed as follows in comparison
to the wild-type pE1(245-961):
Lab-GFP (AUG1/2) 13.4% 1.0%
Lb-GFP (AUG3/4) 1.4% 6.3%
GFP (AUG5) 6.7% 10.4%
21.6% of CAT (100%) 17.7% (82%) of wt activity
The 82% are a sum of all three types of GFP protein forms, initiated from AUA!, AUA2, AUG3, AUA4.
100% is the sum of these products from wild-type pE1(245-961) plasmid having AUG1-4 (Fig. 5B).
BHK-21 cells were infected by recombinant vaccinia virus that expresses T7 polymerase (vTF7-3).