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Title: Biophysical and biochemical studies of the FMDV and EMCV IRES elements
Author: Onuegbu, Anthonia Chinwe
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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The biophysical properties of the hairpin loop structures in the FMDV and EMCV IRES domains three and I, J were studied using UV melting and Circular Dichroism studies. The apical region consists of the hammerhead region that is made up of hairpin loop structures such as the GNRA motifs in the 16mer hairpin loop, (FMDV and EMCV), the heptaloop 15mer in the FMDV and 17mer in the EMCV, The RAAA motif in the 11mer (FMDV and EMCV). The empirical calculations on the folding of the RNA motifs were determined using the Mfold program. The Gibb’s energy obtained was -62.5, -41.9, -19.3, -7.1, -3.3, -5.9 and -2.1 kcal/mol for the 141, 79, 36, 16, 15, 12 and 11mer FMDV RNA motifs, respectively and -10.1, -6.6, -6.8, -14.1, -17.3, and -90.7 kcal/mol for the 16, 17, 28, 39, 44 and 88mer EMCV RNA motifs, respectively, demonstrating the thermodynamic stability of the motifs. The melting temperature of the 141, 79, 36, 16, 15, 12 and 11mer RNA motifs of the FMDV IRES was determined by the first derivative technique. The melting temperature of the 12mer FMDV RNA was the highest with a melting temperature of 78.0 °C signifying it is the most stable followed by the 141, 79, 11, 36, 16, and 15mers FMDV RNA with 76.0, 76.0, 74.0, 72.0, 70.0 and 70.0 °C, respectively. In the EMCV, the 17mer had the highest melting temperature with 78.0 followed by the 16, 88, 28 and 44mers with 77.0, 76.0, 76.0 and 74.0 °C, respectively. The addition of Mg2+ stabilised the RNA motifs. The stabilisation effect on the RNA motifs was not uniform. The melting temperatures obtained were increased by 1.0, 1.0, 4.0, 8.0, 5.0, 2.0 and 2.0 °C for the 141, 79, 36, 16, 15, 12 and 11mer FMDV RNA motifs and 5.0, 4.0, 4.0, 2.0 and 0 °C for the 16, 17, 28, 44 and 88mers EMCV RNA motifs. The most significant effect was observed with the 16mer FMDV, EMCV RNA motifs showing a difference of 8.0, 5.0 °C followed by the FMDV 15mer with a difference of 5.0 °C and EMCV 17mer showing a difference of 4.0 °C. The melting temperature of the EMCV 39mer was not determined without Mg2+, but in the presence of Mg2+ was 76.0 °C. The CD studies showed the RNA motifs to have an A-form conformation with further stabilisation of the loops and stems with the addition of Mg2+.This is the first time the hairpin loop structures in the IRES will be characterised by UV melting and CD studies. Information on the thermal stability of the central domain will provide thermodynamic information on mechanisms of inhibiting protein initiation via the IRES.In addition to the above, the expression and purification of eIF4GcII protein has been carried out.
Supervisor: Ramesh, Vasudevan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available