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Identifying molecular interactions important for PKR function

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posted on 2014-12-15, 10:33 authored by Benjamin William. Gale
Studies involved with the over-expression of full length PKR an d1-170 PKR in-vivo, although successful, did not obtain sufficient quantities of protein for crystallography. Protein studies were continued with the use of in-vitro transcription/translation which showed no PKR inhibitor function in any of the bluetongue viral proteins screened. As well as this, no definitive RNA- kinase interaction was found. Further studies examining the occurrence of a doublet within 1-170 PRK proved that the phenomenon was not the result of residue serine 6 acting as a phosphorylation site or leaky scanning.;RNA binding studies were carried out in order to characterise the dsRNA binding domain of PKR. These studies successfully mapped the region to between PKR residues 10 and 170. Further Poly(I).Poly(C) binding analysis showed the N-terminal boundary to be located to between residues 11 and 30, with the C-terminal boundary between residues 160 and 170. Site directed mutagenesis was undertaken to investigate the contribution of individual amino acids on dsRNA binding within this region. The highly conserved K69 and K159 residues were shown not to be critical for dsRNA binding although a contribution to a positively charged cluster is likely. Mutation of the absolutely conserved A67, A68, A157 and A158 residues did not prevent dsRNA binding or appear to alter the predicted structure of the domain as determined by modelling analysis. Consequently, these conserved residues have been predicted to play a role in maintaining the structure of the domain or more speculatively allowing PKR-PKR dimerisation.

History

Date of award

1997-01-01

Author affiliation

Microbiology

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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