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: both_UTRs_incomplete
The mRNA/+RNA description:
mRNA produced by bicistronic plasmid pFGAL4h-HCV1A/UUG which comprises luciferase and Gal4 genes as the first
and the second cistron, respectively.The full-length 5'-UTR of HCV genomic RNA together with the first 45 nt
of a viral polyprotein gene was inserted in frame into the intercistronic region of the reporter pFGAL4h
vector just behind the 13G:C hairpin, thus making the N-terminal fusion of the first 15 amino acids residues
from the HCV polyprotein with the yeast transcriptional activator Gal4p. The authentic initiation 342AUG codon
of HCV was substituted for 342UUG.
The mRNA/+RNA sequence represented in the +DNA notation:
Credibility of mRNA sequence: end-to-end_sequence_completely_same_as_in_the_experiment
The name of the promoter used to express this mRNA: TPI
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
Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot: unclear_result
Integrity (uniformity) of mRNA tested using RNase protection: not_tested
Integrity (uniformity) of mRNA tested using 5'-RACE: not_tested
Integrity (uniformity) of mRNA tested using primer extension : not_tested
Integrity (uniformity) of mRNA tested using RT-PCR: not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR): not_tested
Integrity (uniformity) of mRNA tested using RNAi: not_tested
Integrity (uniformity) of mRNA tested using S1 nuclease mapping: not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid: not_tested
Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter: not_tested
Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks: not_tested
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:
Plasmid sequence verified (partially/completely) by IRESite (more details in Remarks): plasmid sequence confirmed by IRESite curators by restriction analysis + parts by PCR + sequencing
GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
The total number of notable open-reading frames (ORFs): 2
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
The abbreviated name of this ORF/gene: HCV1A/UUG-GAL4
The description of the protein encoded in this ORF: The fusion protein comprises the first 15 amino acids of HCV viral polyprotein and GAL4 DNA-binding
transcription factor. Gal4p is required for the activation of the GAL genes in response to galactose;
repressed by Gal80p and activated by Gal3p. The authentic initiation codon of HCV was substituted for UUG.
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): 2107-4752
Sc_HCV_type_1a_A/UUG IRES was inserted into intercistronic position of pFGAL4h bicistronic plasmid that has
been designed for the use in the pFGAL4h/PJ69A reporter system. pFGAL4h vector bears genes for the firefly
luciferase and Gal4p as a first and second cistron, respectively. A stable stem-loop hairpin containing 13G:C
pairs was inserted between the two genes in order to prevent ribosome read-through from the first to the
second cistron. The yeast strain PJ69A contains besides the GAL4 and GAL80 deletions also reporter genes
coding for the yeast Ade2 and His3 proteins and for the bacterial beta-galactosidase - all of them under the
control of the Gal4-inducible Gal1 promoter. Thus pFGAL4/PJ69A represents a specialised and sensitive system,
which allows an enhancement of the measured signal by in vivo coupled transcription and enzymatic detection.
This system can be used for both measuring the ratio of the beta-gal/luc enzymatic activities and searching
for IRES activity by screening the colony growth rates on selection media lacking adenine and histidine and
containing various concentrations of the competitive inhibitor of the histidine biosynthetic pathway.
Vector was created by IRESite curator - thus whole sequence is verified by above mentioned methods.
The IRES absolute position (the range includes START and STOP codons or their equivalents): 1712-2096
How IRES boundaries were determined: experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF: 59
3'-end of IRES relative to last base of the STOP codon of the upstream ORF: 443
5'-end of IRES relative to first base of the START codon of the downstream ORF: -395
3'-end of IRES relative to first base of the START codon of the downstream ORF: -11
The sequence of IRES region aligned to its secondary structure (if available):
Remarks:
Sc_HCV_type_1a_A/UUG IRES represents the full-length 5'-UTR of HCV RNA (type 1a) and the first 15 codons of
viral polyprotein. The authentic initiation codon of HCV polyprotein was mutated to UUG.
The relative translation efficiency in % of this IRES: 22.000
Name of IRES used as the positive control: HCV1a
Name of the plasmid used as the positive control. pFGAL4h-HCV1
Name of the plasmid used as the negative control. pFGAL4-L428
IRESite Id of the plasmid used as positive control. 86
IRESite Id of the plasmid used as negative control. 126
The relative translation efficiency in % of the positive control: 100.000
The relative translation efficiency in % of the negative control: 3.800
The size (length) of intercistronic region in the positive control: 453
The size (length) of intercistronic region in the negative control: 432
The effect of 5'-cap analogs on translation: not tested
Rapamycin affects translation: not tested
Type of RNA subject to translation: endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
The relative translation efficiency and size of intercistronic region of negative control have been inserted
as an average values of all negative controls used (pFGAL4-L413,L-428, L456, IDs 126-128).