IRESite_Id:335 characteristics of FMDV_domain_3_GUAA/UCCG IRES studied in pGEM3-FMDV-IRES-domain3

IRESite: The database of experimentally verified IRES structures

The nucleic acid data:

IRESite Id: `335`	Version: `4`
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš	Last change: `2008-02-12 22:23:57`

IRESite record type:

plasmid_with_promoter_and_putative_IRES_without_translational_characterization

The shape of the nucleic acid molecule translated:
linear The quality of the mRNA/+RNA sequence:
only_IRES_fragment

The mRNA/+RNA description:

In vitro T7 run-off transcript used for structure probing experiments of FMDV IRES domain 3 (transcription
runs off at cleaved SmaI site, FMDV IRES domain 3 is between EcoRI and SmaI sites). The GUAA motif
(generalized as GNRA) mutated to UCCG. Please note than an RNA molecule was studied and not its DNA copy.

The mRNA/+RNA sequence represented in the +DNA notation:

gggagaccgg aattcTGTGT TTGGCTCCAC GCTCGATCCA CTGGCGAGTG TTAGTAACAG CACTGTTGCT TCGTAGCGGA GCATGACGGC CGTGGGAACT 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

CCTCCTTGtc cgCAAGGACC CACGGGGCCA AAAGCCACGC CCACACGGGC CCGTCATGTG TGCAACCCCA GCACGGCGAC TTTACTGCGA AACCCACTTT 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

AAAGTGACAT TGAAACTGGT ACCCACAccc
^201       ^211       ^221

Credibility of mRNA sequence:

end-to-end_sequence_reverse_engineered_and_should_match_experiment

The name of the plasmid:
pGEM3-FMDV-IRES-domain3_GUAA/UCCG 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:
FMDV

The origin of IRES in the plasmid:

viral

The donor organism of the IRES segment:

Foot-and-mouth disease virus C-S8c1

The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:

tatagtgtca cctaaatcgt atgtgtatga tacataaggt tatgtattaa ttgtagccgc gttctaacga caatatgtac aagcctaatt gtgtagcatc 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

tggcttactg aagcagaccc tatcatctct ctcgtaaact gccgtcagag tcggtttggt tggacgaacc ttctgagttt ctggtaacgc cgttccgcac 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

cccggaaatg gtcagcgaac caatcagcag ggtcatcgct agccagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

tggccggggg actgttgggc gccatctcct tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

tcgcataagg gagagcgtcg atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 
^1401      ^1411      ^1421      ^1431      ^1441      ^1451      ^1461      ^1471      ^1481      ^1491      

tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 
^1501      ^1511      ^1521      ^1531      ^1541      ^1551      ^1561      ^1571      ^1581      ^1591      

aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 
^1601      ^1611      ^1621      ^1631      ^1641      ^1651      ^1661      ^1671      ^1681      ^1691      

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 
^1701      ^1711      ^1721      ^1731      ^1741      ^1751      ^1761      ^1771      ^1781      ^1791      

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 
^1801      ^1811      ^1821      ^1831      ^1841      ^1851      ^1861      ^1871      ^1881      ^1891      

tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 
^1901      ^1911      ^1921      ^1931      ^1941      ^1951      ^1961      ^1971      ^1981      ^1991      

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 
^2001      ^2011      ^2021      ^2031      ^2041      ^2051      ^2061      ^2071      ^2081      ^2091      

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 
^2101      ^2111      ^2121      ^2131      ^2141      ^2151      ^2161      ^2171      ^2181      ^2191      

cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 
^2201      ^2211      ^2221      ^2231      ^2241      ^2251      ^2261      ^2271      ^2281      ^2291      

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 
^2301      ^2311      ^2321      ^2331      ^2341      ^2351      ^2361      ^2371      ^2381      ^2391      

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 
^2401      ^2411      ^2421      ^2431      ^2441      ^2451      ^2461      ^2471      ^2481      ^2491      

tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 
^2501      ^2511      ^2521      ^2531      ^2541      ^2551      ^2561      ^2571      ^2581      ^2591      

tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 
^2601      ^2611      ^2621      ^2631      ^2641      ^2651      ^2661      ^2671      ^2681      ^2691      

agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggctt atcgaaatta atacgactca ctatagggag 
^2701      ^2711      ^2721      ^2731      ^2741      ^2751      ^2761      ^2771      ^2781      ^2791      

accggaattc TGTGTTTGGC TCCACGCTCG ATCCACTGGC GAGTGTTAGT AACAGCACTG TTGCTTCGTA GCGGAGCATG ACGGCCGTGG GAACTCCTCC 
^2801      ^2811      ^2821      ^2831      ^2841      ^2851      ^2861      ^2871      ^2881      ^2891      

TTGtccgCAA GGACCCACGG GGCCAAAAGC CACGCCCACA CGGGCCCGTC ATGTGTGCAA CCCCAGCACG GCGACTTTAC TGCGAAACCC ACTTTAAAGT 
^2901      ^2911      ^2921      ^2931      ^2941      ^2951      ^2961      ^2971      ^2981      ^2991      

GACATTGAAA CTGGTACCCA CAcccgggga tcctctagag tcgacctgca ggcatgcaag cttgtattc
^3001      ^3011      ^3021      ^3031      ^3041      ^3051      ^3061

GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:

The total number of notable open-reading frames (ORFs):

0

Citations:

Martinez-Salas E., Saiz J. C., Davila M., Belsham G. J., Domingo E. (1993) A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo. J. Virol. 67(7):3748-3755

Additional data: http://bccm.belspo.be/db/lmbp_plasmid_details.php?NM=pBIC

Ramos R., Martinez-Salas E. (1999) Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES). RNA. 5(10):1374-1383

IRESs:

IRES:

Version: `2`	Last change: `2008-02-12 20:16:16`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš
The IRES name: `FMDV_domain_3_GUAA/UCCG`	The functional status of IRES: `defective`

The IRES absolute position (the range includes START and STOP codons or their equivalents):

15-229

How IRES boundaries were determined:
experimentally_determined

The sequence of IRES region aligned to its secondary structure (if available):

cTGTGTTTGG CTCCACGCTC GATCCACTGG CGAGTGTTAG TAACAGCACT GTTGCTTCGT AGCGGAGCAT GACGGCCGTG GGAACTCCTC CTTGtccgCA 
^15        ^25        ^35        ^45        ^55        ^65        ^75        ^85        ^95        ^105       

AGGACCCACG GGGCCAAAAG CCACGCCCAC ACGGGCCCGT CATGTGTGCA ACCCCAGCAC GGCGACTTTA CTGCGAAACC CACTTTAAAG TGACATTGAA 
^115       ^125       ^135       ^145       ^155       ^165       ^175       ^185       ^195       ^205       

ACTGGTACCC ACAcc
^215       ^225

Citations:

Fernandez-Miragall O., Martinez-Salas E. (2003) Structural organization of a viral IRES depends on the integrity of the GNRA motif. RNA. 9(11):1333-1344

Regions with experimentally determined secondary structures:

A region with the experimentally determined secondary structure:

IRESite 2D Struct Id: 12

Version: `2`	Last change: `2008-02-12 20:16:16`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš
The function of the 2D structure: `IRES`	The 2D structure causes frameshift: `unknown`

The absolute position of the experimentally mapped region (the range includes START and STOP codons or their equivalents):
33-213

The underlying nucleic acid sequence and structure of the mapped region:

TCGATCCACT GGCGAGTGTT AGTAACAGCA CTGTTGCTTC GTAGCGGAGC ATGACGGCCG TGGGAACTCC TCCTTGtccg CAAGGACCCA CGGGGCCAAA 
((.((..((( ((((((((.( (.((((((.. ...((((((( ......)))( (((((((((( ((((...... (((((..... .))))))))) )))(((.... 
^33        ^43        ^53        ^63        ^73        ^83        ^93        ^103       ^113       ^123       

AGCCACGCCC ACACGGGCCC GTCATGTGTG CAACCCCAGC ACGGCGACTT TACTGCGAAA CCCACTTTAA AGTGACATTG A
.)))..(((. .....))))) ))))))(... .......... ..)))))... ..)))))))) )))))))).. )))...)).) )
^133       ^143       ^153       ^163       ^173       ^183       ^193       ^203       ^213

There is no Vienna RNA package installed on the server or some error/warning messages were output. Due to that maybe we cannot prepare 2D structures for display. The error/warning message was:

WARNING: bases 13 and 166 (CU) can't pair!
WARNING: bases 15 and 164 (AA) can't pair!
WARNING: bases 17 and 162 (UC) can't pair!
WARNING: bases 21 and 159 (AA) can't pair!
WARNING: bases 24 and 157 (AG) can't pair!
WARNING: bases 25 and 156 (AC) can't pair!

STDOUT was:

UCGAUCCACUGGCGAGUGUUAGUAACAGCACUGUUGCUUCGUAGCGGAGCAUGACGGCCGUGGGAACUCCUCCUUGuccgCAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGUCAUGUGUGCAACCCCAGCACGGCGACUUUACUGCGAAACCCACUUUAAAGUGACAUUGA
((.((..(((((((((((.((.((((((.....(((((((......)))(((((((((((((((......(((((......))))))))))))(((.....)))..(((......)))))))))))(...............))))).....))))))))))))))))..)))...)).)) (-34.60)

Rendering structure of FMDV IRES in an artificial transcript 181 nt long with energy of -34.60 kcal/mol as calculated by RNAeval using VARNA Java applet with some IRESite improvements (see VARNA modified by IRESite). Hold left mouse button to move structure parts, hold right mouse button to move whole structure, use mouse wheel to zoom. Right mouse-click opens a menu to export into JPG/SVG and many other options.

You need a Java-enabled browser so that modified varsion of VARNA could be started. See http://www.iresite.org/VARNA/ for more details.

Remarks:

FMDV IRES domain3 with GUAA to UCCG mutation as shown in Fig. 8A

3.1.1. Enzymes used to characterize at least partially the 2D structure.