The nucleic acid data:
IRESite Id: 280 Version: 1
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-06-04 21:59:34
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 first 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 name of the plasmid:
pE1(245-961)deltaA1
The name of the promoter used to express this mRNA:
  T7
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  not tested
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  not tested
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
ERAV
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Equine rhinitis A virus 1 393/76
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pE1(245-961)deltaA1.jpg
The total number of notable open-reading frames (ORFs):
  5
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
CAT
The description of the protein encoded in this ORF:
chloramphenicol acetyltransferase
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  615-1274
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lab-GFP
The description of the protein encoded in this ORF:
green fluorescent protein extended at its N-terminus by 39 aminoacid residues encoded by 3'-end of ERAV IRES and partly by multiple cloning sequence site (as if initiated from AUG2).
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):
  1901-2821
ORF
ORF position:   3
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lb-GFP
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
ORF
ORF position:   4
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Lb-GFP
The description of the protein encoded in this ORF:
green fluorescent protein extended at its N-terminus by 18 aminoacid residues encoded by 3'-end of ERAV IRES and partly by multiple cloning sequence site (as if initiated from AUG4)
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):
  1964-2821
ORF
ORF position:   5
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
GFP
The description of the protein encoded in this ORF:
green fluorescent protein
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):
  2018-2821
Remarks:
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 was mutated to AUA (Fig. 5A).
Citations:
Hinton T. M., Li F., Crabb B. S. (2000) Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. J. Virol. 74(24):11708-11716
IRESs:
IRES:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  ERAV_245-961_deltaA1
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:
  -611
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  103
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
This mutation confirms that Lab-GFP was synthesized mostly from AUG1 so the IRES is still largely functional.
Citations:
Hinton T. M., Li F., Crabb B. S. (2000) Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. J. Virol. 74(24):11708-11716
The translation experiments:
Translation results:
IRESite Translation Id: 358
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens BHK-21 (ATCC CCL-10)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  87.000
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
Remarks:
Mutation of 1st 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):

                     wt                 pE1(245-961)deltaA1
Lab-GFP (AUG1/2)  13.4%                 11.8%
 Lb-GFP (AUG3/4)   1.4%                  1.3%
    GFP (AUG5)     6.7%                  5.6%
_____________________________________________
          21.6% of CAT (100%)           18.7% (87%) of wt activity


The 87% are a sum of all three types of GFP protein forms, initiated from AUG2, AUG3, AUG4.
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).
Citations:
Hinton T. M., Li F., Crabb B. S. (2000) Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. J. Virol. 74(24):11708-11716
Last change to the database: 2015-04-16 16:45:23 GMT+1